Claire Objois
email: claire.objois@gmail.com

Sarah Gebeile-Chauty

Is premature birth an orthodontic risk factor? A controlled epidemiological clinical study

Keywords:

Introduction > Although a "catch-up" growth occurs in early childhood, respiratory system immaturity and the use of oral and naso-gastric probes for prematurely born children are responsible for palatal and cranio-facial alterations. Objective > To assess whether prematurity is a risk factor in orthodontics in terms of malocclusions, dysmorphoses and orthodontic treatment need compared to term-born children. To compare the differences of risk factors among these preterm birth subtypes according the severity. Materials and methods > Distributed questionnaires within an orthodontic practice and in the Department of Odontology, Lyon Hospital, France. The evaluation criteria were calculated from the casts, photographs and radiographies carried out during the initial assessment. A statistical intergroup comparison was performed. Results > Of the 537 questionnaires obtained, 47 preterm patients and 150 term-born patients were included in our study. Preterm infants had significantly more bilateral crossedocclusions than patients in the term group (p = 0.003). In addition, very preterm and extremely preterm children had significantly more impacted tooth (p = 0.049) and a higher Index of Orthodontic Treatment Need (IOTN grade 5, p = 0.003) compared with term children. Conclusion > Very preterm and extremely preterm births (occurring more than 8 weeks before the term) represent a risk factor in orthodontics and, therefore, a public health problem.

Introduction

According to the World Health Organization (WHO), prematurity is defined by a birth occurring before 37 completed weeks of gestation (i.e. more than 4 weeks before the term). Three subgroups can be distinguished [START_REF] Lacroze | Prématurité : définitions, épidémiologie, étiopathogénie, organisation des soins[END_REF]: moderately preterm birth (33 to 36 +6days weeks); very preterm birth (28 to 32 +6days weeks); extremely preterm birth (<28 weeks). Prematurity affects around 15 million children a year (11% of live births), 60,000 of them in France, and its incidence has been increasing since the 1990s according to the WHO. More than 60% of premature births occur in Africa and South Asia, but this is a global problem. In Europe, the 2004 EuroPeristat data [2] shows a preterm birth rate between 5 and 11% of live births. France is rather in the low range with a prematurity rate of 6.3% (6,4% to 7.0% according to the AUDIPOG sentinel network (Association des Utilisateurs de Dossiers Informatisés en Périnatologie, Obstétrique et Gynécologie), [START_REF]La santé périnatale en 2004-2005[END_REF] in 2007. In the case of very preterm births (22 to 32 weeks +6days ), the MOSAIC cohort [START_REF] Zeitlin | Differences in rates and short-term outcome of live births before 32 weeks of gestation in Europe in 2003: results from the MOSAIC cohort[END_REF] and EuroPeristat (European perinatal Heath report statistics) [2] reported an overall rate of 13.2/1000 births, representing 14.3% of total premature births. Less than 5% of preterm infants have a gestational age below 28 weeks (extremely preterm birth) [START_REF] Lacroze | Prématurité : définitions, épidémiologie, étiopathogénie, organisation des soins[END_REF]. The incidence of prematurity in France has been constantly increasing. Multiple factors are involved: increased number of premature triggered births, increased maternal age, increased use of medically assisted procreation and multiple pregnancies resulting from these techniques. Besides, the improvement of neonatal resuscitation techniques allows a greater survival of new-borns who are increasingly immature.

Characterized by a global immaturity, a preterm infant's body is not able to function and to defend itself. First, the immature lungs of the preterm infant sometimes require artificial oxygen supply (oxygen therapy) and assisted ventilation. Although it's vital, this oxygenation aid is not without risk, especially for infants born under 32 weeks. They are prone to develop chronic respiratory failure (bronchopulmonary dysplasia) when the oxygen requirement persists beyond 28 days of life. This respiratory pathology aggravates the eating disorders linked to difficulties in the coordination of sucking-swallowing-breathing and more generally to digestive immaturity. In addition, as the maturation of sucking occurs only at the end of the normal term of gestation, the baby born before 34 weeks of age is unable to feed on his own [START_REF] Haddad | La prise en charge orthophonique du bébé prématuré en service de néonatalogie[END_REF]. Feeding through an oral or nasogastric tube will then be necessary. This type of nutritional assistance leads to harmful consequences at several levels, irritation of the nose and/or the pharynx, or a disturbance of the sucking-swallowing reflex. It is also important to note the morphogenetic role of foetal sucking and swallowing on the development of the oral cavity [START_REF] Couly | Sucking, a qualitative index of neonatal maturity[END_REF].

Justification of the study

As other tissues and organs of the body, oral structures are affected by premature birth [START_REF] Paulsson | A systematic review of the consequences of premature birth on palatal morphology, dental occlusion, tooth-crown dimensions, and tooth maturity and eruption[END_REF]. Premature birth and its adaptations have an effect on occlusal development, which may be associated with an increased orthodontic treatment need [START_REF] Zaidi | Preterm Birth: A Primary Etiological Factor for Delayed Oral Growth and Development[END_REF]. Moreover, palate morphology alterations, related to the use of probes, have been described [9][START_REF] Kopra | Prevalence of oral defects among neonatally intubated 3-to 5and 7-to 10-year old children[END_REF][START_REF] Macey-Dare | Long-term effect of neonatal endotracheal intubation on palatal form and symmetry in 8-11-year-old children[END_REF]. While early catch-up growth periods seem to mitigate these defects [START_REF] Niklasson | Growth in very preterm children: a longitudinal study[END_REF], the head circumference is smaller at birth and remains lower with no catch-up growth until the age of 11 [START_REF] Farooqi | Growth in 10-to 12-year-old children born at 23 to 25 weeks' gestation in the 1990s: a Swedish national prospective follow-up study[END_REF]. The craniofacial morphology of young patients (8-10 years) born extremely premature is characterized by a shorter anterior cranial base, a less convex profile and a shorter maxillary length [START_REF] Paulsson | Malocclusion traits and orthodontic treatment needs in prematurely born children[END_REF]. The idea that prematurity is a risk factor in orthodontics is currently not yet proven. Indeed, the question of the impact of prematurity on orthodontic malocclusions and the orthodontic treatment need in these patients remains seldom studied. Only a few Scandinavian studies [START_REF] Paulsson | A systematic review of the consequences of premature birth on palatal morphology, dental occlusion, tooth-crown dimensions, and tooth maturity and eruption[END_REF][START_REF] Paulsson | Malocclusion traits and orthodontic treatment needs in prematurely born children[END_REF][START_REF] Paulsson | Craniofacial morphology in prematurely born children[END_REF][START_REF] Harila-Kaera | Permanent tooth crown dimensions in prematurely born children[END_REF][START_REF] Harila-Kaera | Sagittal occlusal relationships and asymmetry in prematurely born children[END_REF][START_REF] Harila | Open bite in prematurely born children[END_REF][START_REF] Rythén | Dento-alveolar characteristics in adolescents born extremely preterm[END_REF] have analysed the prevalence of malocclusions in premature subjects compared to term-born children, but the conclusions are variable and even contradictory as regards to the malocclusions most frequently observed. Because of contradictory results and a lack of longitudinal studies, scientific evidence is too weak to link prematurity with malocclusions [START_REF] Paulsson | A systematic review of the consequences of premature birth on palatal morphology, dental occlusion, tooth-crown dimensions, and tooth maturity and eruption[END_REF] : low number of recent studies related to this topic, lack of sufficient scientific evidence, contradictions of the obtained results, the variability of the methodologies and old studies, can no longer be used as a reference because the care protocols evolve, without counting that they may vary from one country to another. Moreover, no French study has been carried out for now on this subject. The studies found are all Scandinavian (Swedish and Finnish), but there are significant differences between countries in terms of malocclusion prevalence and orthodontic treatment need [START_REF] Souames | Orthodontic treatment need in French schoolchildren: an epidemiological study using the Index of Orthodontic Treatment Need[END_REF]. The large variability according to the countries in the preterm infants care and the difference of preterm birth proportion in the populations prevents us from extrapolating these results on the French population. The main objective of this study is to assess whether prematurity is a risk factor in orthodontics, in terms of malocclusions, dysmorphoses and treatment need. The secondary objective is to compare, according to these same criteria, the different stages of prematurity (extremely / very preterm birth and moderately premature).

Materials and methods

Specificity and study place

This is a cross-sectional comparative epidemiological study. Patients were recruited at the orthodontic practice of doctors Sarah CHAUTY and Marie-José GOUMY in the east of Lyon (56 Rue de la République / 69150 DECINES CHARPIEU, France), and in the orthodontic unit of the Consultations and Dental Treatments Service (6-8, place Depéret / 69007 LYON, France).

Study population

Recruitment method Over a period of 4 months, a questionnaire was systematically distributed to all patients during their visit to the orthodontic practice. Parents were asked whether their child was born prematurely (at least 4 weeks ahead of the term or before 37 weeks) or not. If the child was premature, they were asked to specify the child's weight at birth, the number of weeks of gestation and the number of weeks of advance before the date of the intended term. For all patients, we asked if they allowed us to study the orthodontic record, the photos and radiographs of the child taken at the time of the initial orthodontic examination. Questionnaires that were not correctly completed or did not allow participation in the study were discarded. On the other hand, when the number of weeks in advance was not certainly known by the parent, or when there was a doubt, the patient was not included in the study. We established two groups of patients, one group of premature (P) and a control one (C) of term-born children. Two subgroups were established within the "premature" group (P = A + B): group A comprising extremely and very preterm children, born < 33 weeks and group B consisting of moderately preterm children born ≥ 33 and ≤ 37 weeks.

Inclusion Criteria

All patients in the study had to be between 6 and 16 years old at the time of the initial orthodontic examination ; they were not to have had a previous orthodontic treatment; they were asked to complete the questionnaire and give the permission to use their medical documents for the study; they had to have performed at least one orthodontic examination with diagnosis, treatment plan and complete a workable file containing all documents related to the study: photographs, casts, initial radiographs (panoramic radiography and lateral teleradiography).

Non-Inclusion Criteria

Not included in the study were patients with congenital syndrome or pathology, failure to correctly complete the questionnaire, no willingness to participate in the study, orthodontic history, and incomplete orthodontic records.

Evaluation criteria

Orthodontic Treatment Need is assessed through the IOTN Dental Health Component (IOTN Index Of Treatment Need). Patient abnormalities are investigated and only the most severe anomaly is considered and determines the score (from 1 to 5): Grades 1 and 2: no orthodontic treatment need, Grade 3: moderate treatment need, Grades 4 and 5: proven orthodontic treatment need.

Malocclusions

The examination of the malocclusions is carried out from the endobuccal photographs and the standardized photographs of the plaster casts, as well as from the profile radiography.

-Determination of the Angle Class: Angle Class I (maxillary canine tip in the embrasure between the canine and the first mandibular premolar), Angle Class II (if distoclusion of the mandibular molar greater than ½ cusp), Angle Class III (if mesioclusion of the mandibular molar greater than ½ cusp).

-Millimetric evaluation of the overjet and the overbite (carried out on the profile radiography), with respect to the plane of occlusion (middle of the molar covering -middle of the incisal covering). We consider that the patient has an anterior supraclusion if the overbite is greater than 4 mm, and an infraclusion if it is less than 0.

-Determination of the presence or absence of anterior/posterior crossbite, uni or bilateral: Anterior, if 1, 2, 3 or 4 incisors are in linguoclusion ; Posterior, if 1 or more teeth is inverted in articulation. A crossbite is considered beyond the "cuspid-cuspid" relationship in the transverse dimension.

-Evaluation of the presence of diastema/crowding (according to the index of Little) to the mandibular arch (from the distal face of the 33 to the distal face of the 43) :

The crowding is considered important if the value is greater than the mesio-distal diameter of the patient central mandibular incisor. We record the presence of diastema when these are greater than half diameter of the mandibular incisor. -Evaluation of the major rotations (> 30 ° with respect to the ideal arch line) -Presence or absence of agenesis, supernumerary tooth, transposition -Highlighting of impacted teeth thanks to the panoramic examination The examination was carried out by the same examiner. The reproducibility of the observations was checked on a sample of patients by a second examiner.

Cephalometry / Dysmorphoses

We computed (using Ortholeader © software) angular and metric values obtained from the Tweed cephalometric analysis : skeletal values: sagittal: SNA, SNB, ANB, AO-BO; vertical : FMA dental values: IMPA, I / F Indication of treatment for transverse deficiencies We identified the indications of transpalatal distractor, quad'helix or an expansion palatal plate.

Statistics

Statistical tests (Chi2 or Chi square-test and Student test) were carried out in order to compare the different groups and subgroups, in terms of malocclusions, dysmorphoses and treatment need. For all quantitative variables, the average and the standard deviation were calculated. For the qualitative variables, data were transcribed as a percentage. For the quantitative response variables -cephalometric values (SNA, SNB, ANB, AOBO, FMA, IMPA, I/F), overbite, overjet -comparisons between the groups were performed using the Student test; as for the nominal qualitative response variables (malocclusions, IOTN grades, prescription of the rapid maxillary expansion) the Chi2 test was performed. The threshold of significance was 5%.

Results

Description of study population

The diagram of the study is presented in Figure 1. 537 questionnaires were completed by the parents/accompanying of patients in the offices of doctors CHAUTY and GOUMY. Among the questionnaires collected, 49 indicated that the child was born prematurely (born more than 4 weeks in advance). We randomly selected 150 patients with a complete orthodontic record (initial endobuccal photographs, standardized casts, lateral teleradiography and panoramic examination before treatment) to form the control group (Group C). Following the same protocol and completing the same questionnaire, 5 additional premature children, treated in orthodontics at the Department of Odontology in Lyon Hospital, could be included in the study. Of the 54 premature infants, 7 were incomplete (X-rays, photographs or missing casts) and could not be exploited as part of the study. In the end, 47 children born premature were able to be definitively included, constituting the "premature" group (group P).

The first subgroup, including "extremely and very preterm" patients born < 33 weeks (group A), is composed of 13 patients. The second subgroup, consisting of "moderate" preterm infants (group B), is made of 34 patients. The table I compares the age and sex of the children by group. There was no statistically significant difference between the two groups with regard to the age and distribution of girls and boys (p> 0.05).

Inter-group statistical comparison

Analysis of orthodontic malocclusions (table II)

Premature versus control : We found a significant difference between preterm infants and the control group for bilateral posterior crossbite (p <0.05), which are found more frequently in preterm infants. Subgroups comparison (Extremely/very/moderately preterm) : By comparing more specifically the extremely and very preterm subgroup (A) to the control group, we also find a significant difference in the presence of inclusions, retentions, and enclosed teeth. There was no significant difference between very and moderately preterm infants or between preterm infants and control group for any studied clinical values.

Skeletal and dental cephalometric values analysis (table III)

No significant difference was found between the preterm group and the control group, and no difference between the different premature subgroups, for none of these values (p> 0.05). Indication of treatment for transverse deficiencies 15 out of the 47 premature patients had maxillary transverse expansion, or approximately 27%, and 40 out of 150 patients in the control group (32%). The difference is not statistically significant.

Treatment need / IOTN analysis (tables IV and V)

The results of our study revealed a greater orthodontic treatment need in the preterm group compared to the control group : 62.5% (A + B) versus 52% (C) considering grade 4 and 5 only (high treatment need) 81.3% (A + B) versus 68.7% (C), adding grade 3 to grades 4 and 5 A statistically significant difference was found for grade 5 between the extremely and very preterm group (A) compared to the control group (C) (53.3% versus 16.7%).

Discussion

Description of the study population

In our study, 9.1% of the patients surveyed were born prematurely. In comparison with the EuroPeristat data [2] with a preterm percentage in France of 6.3% in 2004 (7.0% according to the sentinel network AUDIPOG [START_REF]La santé périnatale en 2004-2005[END_REF]), it appears that our percentage is slightly higher. In addition, 13% of the preterm patients in our study were extremely preterm infants and 15% were very preterm infants. These percentages are, again, higher than those found in the epidemiological studies cited in the first part. This could be explained by the fact that the population studied is an orthodontic population. Premature babies with an increased need for treatment compared with the term population, could be overrepresented in the orthodontic population (compared to the general population). However, the number of participants in the groups A and B remain low and would require other investigations on a larger population.

Study protocol

In this retrospective study, the decision to prescribe a treatment of the transverse dimension was made by "blind" practitioners, without knowing whether the patient was premature or not and was therefore not influenced.

In addition, traces and cephalometric analyses were also carried out blindly and by the same examiner. They were executed before knowing to which group the patient belonged, so there was no overvaluation of the measured values. Regarding the need for treatment, we chose to use the IOTN (Index Of Treatment Need) dental health component (or DHC) in our study. This method is widely used in the literature, simple, valid, rapid and reproducible to evaluate the orthodontic treatment need of a population [START_REF] Beglin | A comparison of the reliability and validity of 3 occlusal indexes of orthodontic treatment need[END_REF][START_REF] Uçüncü | The use of the Index of Orthodontic Treatment need (IOTN) in a school population and referred population[END_REF][START_REF] So | A comparative study using the Occlusal Index and the Index of Orthodontic Treatment Need[END_REF].

Malocclusions and dysmorphoses

Intra-arch abnormalities

Prematurity is thus often associated with a transverse deficiencies of the maxilla and a decrease of the maxillary and mandibular arch perimeters in relation to the dental volume and the necessary space on the arch. It results in a lack of space for the development of the teeth, responsible for dental retention and impactions or more anarchic eruptions with dental malpositions, ectopias and dental rotations. Diastemas Paulsson et al. [START_REF] Paulsson | Malocclusion traits and orthodontic treatment needs in prematurely born children[END_REF] found significantly more diastemas in preterm infants (47.9% versus 29.3% in the control group). In our study, we found a slightly higher percentage in premature versus control patients, but this difference is too low to be significant. This difference may be explained by the variation in the calculation method between the two studies. Indeed, we have calculated the crowding and diastemas only at the mandibular arch (according to the index of Little) and not at the two arches, but the diastemas are more frequently found in the maxilla. Crowding Furthermore, Paulsson et al. [START_REF] Paulsson | Malocclusion traits and orthodontic treatment needs in prematurely born children[END_REF], like us, obtain a greater prevalence of crowding situations in the case of prematurity, but in both cases the difference is not significant.

Impactions retentions, retained teeth

We have demonstrated significantly more dental inclusions and retention in extremely and very preterm infants. Paulsson et al. [START_REF] Paulsson | Malocclusion traits and orthodontic treatment needs in prematurely born children[END_REF] did not analyse inclusions and teeth retentions in their study, but they frequently found more ectopic teeth (canines and molars) in premature infants (not significant).

Inter-arch abnormalities Transverse dimension

Crossbite

In our study, we found a significantly higher percentage of bilateral posterior crossbite in the preterm group (P) than in the control group (C) (10.6% vs. 0.7%, p = 0.003). This malocclusion may be related to the sometimes-prolonged use of the nasogastric tube in the neonatal period and the disruption of oral functions (sucking-swallowing and ventilation) described in premature infants. These dysfunctions may persist over the long-term and affect the growth and normal development of the palate [START_REF] Couly | Sucking, a qualitative index of neonatal maturity[END_REF]. Kopra and Davis [10] also found a significantly higher crossbite rate in intubated patients. Other authors did not find any significant difference between premature and control groups [START_REF] Macey-Dare | Long-term effect of neonatal endotracheal intubation on palatal form and symmetry in 8-11-year-old children[END_REF][START_REF] Paulsson | Malocclusion traits and orthodontic treatment needs in prematurely born children[END_REF][START_REF] Fadavi | The oral effects of orotracheal intubation in prematurely born preschoolers[END_REF].

Palate dimensions

The width of the palate would be lower in premature infants who had been intubated [START_REF] Kopra | Prevalence of oral defects among neonatally intubated 3-to 5and 7-to 10-year old children[END_REF][START_REF] Macey-Dare | Long-term effect of neonatal endotracheal intubation on palatal form and symmetry in 8-11-year-old children[END_REF], but this difference would disappear after 11 years [9,19] as if the initial lag tends to normalize.

Palatal abnormalities

Infant bone is extremely malleable and easily deformed under the pressure of the naso or oropharyngeal catheter or following a direct trauma of the laryngoscope [START_REF] Angelos | Oral complications associated with neonatal oral tracheal intubation: a critical review[END_REF]. Some authors find a positive correlation between intubation and morphological alterations of the palate and upper alveolar contour [START_REF] Alves | The influence of orotracheal intubation on the oral tissue development in preterm infants[END_REF] such as asymmetric grooves and a deep palatal arch [START_REF] Kopra | Prevalence of oral defects among neonatally intubated 3-to 5and 7-to 10-year old children[END_REF][START_REF] Macey-Dare | Long-term effect of neonatal endotracheal intubation on palatal form and symmetry in 8-11-year-old children[END_REF][START_REF] Fadavi | The oral effects of orotracheal intubation in prematurely born preschoolers[END_REF][START_REF] Procter | Deformation of the palate in preterm infants[END_REF][START_REF] Cortines | Associated factors and persistence of palatal groove in preterm infants: a cohort study[END_REF], which may persist after age of 10 years [START_REF] Kopra | Prevalence of oral defects among neonatally intubated 3-to 5and 7-to 10-year old children[END_REF][START_REF] Macey-Dare | Long-term effect of neonatal endotracheal intubation on palatal form and symmetry in 8-11-year-old children[END_REF]. Palate growth and remodelling would "repair", for other authors, the deformities resulting from the intubation process, as no significant difference could be found in the long term [START_REF] Paulsson | A systematic review of the consequences of premature birth on palatal morphology, dental occlusion, tooth-crown dimensions, and tooth maturity and eruption[END_REF]9,[START_REF] Seow | Oral complications of premature birth[END_REF][START_REF] Seow | Effect of neonatal laryngoscopy and endotracheal intubation on palatal symmetry in two-to five-year old children[END_REF]. We did not analyse the influence of neonatal intubation in our study, as it has been studied in previous studies [START_REF] Kopra | Prevalence of oral defects among neonatally intubated 3-to 5and 7-to 10-year old children[END_REF][START_REF] Macey-Dare | Long-term effect of neonatal endotracheal intubation on palatal form and symmetry in 8-11-year-old children[END_REF]. It would be interesting in the future to establish two subgroups within the premature population in order to assess whether this transverse deficiency is the consequence of immaturity alone or should be more imputed to the action of the oral probe. However, we know that in most of the cases, extremely and very preterm neonates are intubated and this factor probably explains the difference in terms of bilateral cross occlusions that we obtained between the two subgroups A and B.

Vertical dimension

Supraclusion

We did not show a significant difference between preterm and non-preterm patients in regards to supraclusion. For Paulsson et al. [START_REF] Paulsson | Malocclusion traits and orthodontic treatment needs in prematurely born children[END_REF], supraclusion prevalence was significantly higher in premature infants (EPT and VPT) than in the control group and was the most common type of malocclusion found in premature infants. For Rythén et al. [START_REF] Rythén | Dento-alveolar characteristics in adolescents born extremely preterm[END_REF], this is the second most common malocclusion after class II. We found on average more supraclusions in our control term-born population compared to the non-premature populations of these two Swedish studies [START_REF] Paulsson | Malocclusion traits and orthodontic treatment needs in prematurely born children[END_REF][START_REF] Rythén | Dento-alveolar characteristics in adolescents born extremely preterm[END_REF]. This can be explained by the fact that our "control" population is an orthodontic and not a general population. Moreover, it would be interesting to study the supraclusion prevalence in Sweden and to compare it with the French one. However, we did not find any article describing Swedish malocclusion prevalences. Anterior open-bite Harila et al. [START_REF] Harila | Open bite in prematurely born children[END_REF] demonstrated a higher prevalence of anterior infraclusion in prematurely born children, which was not demonstrated in our study. Premature babies are more likely to be influenced by the etiological factors that influence the development of an anterior infraclusion: respiratory infections, inadequate nasal/oral breathing, oral habits and other medical problems.

Sagittal dimension

In our study, no conclusion could be drawn concerning the sagittal dimension (from a dental and skeletal point of view). Rythén et al. [START_REF] Rythén | Dento-alveolar characteristics in adolescents born extremely preterm[END_REF] found that Class II malocclusion is twice as common in premature infants (30% in EPT versus 15% in the control group). Harila-Kaera et al. [START_REF] Harila-Kaera | Sagittal occlusal relationships and asymmetry in prematurely born children[END_REF] noted a significantly higher prevalence of canine class III in the preterm group. The prevalence of mesial molar occlusion was greater in the preterm group and significantly more bilateral class I was found in preterm infants and less class II compared to the control group. We observed that in our control group we counted many more cases of class II, compared to these two Scandinavian studies [START_REF] Paulsson | Malocclusion traits and orthodontic treatment needs in prematurely born children[END_REF][START_REF] Rythén | Dento-alveolar characteristics in adolescents born extremely preterm[END_REF]. This difference can be explained by the fact that our study population was recruited from an orthodontic practice, which was not the case in other studies. In addition, class II malocclusion is particularly common in France, according to the HAS [START_REF]Bilan de dysmorphose dento-maxillo-faciale[END_REF]. Cephalometry Regarding the evaluation of cephalometric values, we found no significant difference between the different groups, or according to the different prematurity degrees. A significantly shorter anterior cranial base and a less convex skeletal profile have been found in the literature in extremely premature infants [START_REF] Paulsson | Craniofacial morphology in prematurely born children[END_REF]. A significantly shorter length of the maxilla was noted in the 2 groups (VPT and EPT) compared to the control group. In the same study, mandibular incisors were also significantly more linguoverted in extremely premature patients (EPT). These elements thus reflect a class III skeletal profile, more commonly found in extremely and very preterm infants. The results of our study do not lead to the same conclusions. This is certainly due to the age of the patients in the study : the subjects of Paulsson et al. [START_REF] Paulsson | Craniofacial morphology in prematurely born children[END_REF] were significantly younger than in our study, so a significant anteroposterior maxillary growth could ensure catch-up leading to adolescence at a similar size [START_REF] Laowansiri | Maxillary growth and maturation during infancy and early childhood[END_REF].

Indication of treatment for transverse deficiencies

This evaluation criteria has not been studied in previous studies. We do not find in our study any significant difference between the different groups.

Treatment Need (IOTN)

In our study, we found a significantly higher percentage of "grade 5" in the extremely and very preterm group than in the term group (53.9% versus 16.7%, p = 0.003). The high percentage of grade 5 can be explained in extremely and very preterm babies by the very high number of situations of dental inclusion or retention (7 of 13) or transposition (8 out of 13), corresponding to a grade 5 according to the IOTN. We can thus conclude that extremely and very preterm birth lead to a higher orthodontic treatment need, in relation to malocclusions more frequently observed in these patients. The enamel defects described in the literature (hypoplasia) could participate in the difficulties of natural placement of teeth [START_REF] Jacobsen | Developmental enamel defects in children born preterm: a systematic review[END_REF]. Paulsson et al. [START_REF] Paulsson | Malocclusion traits and orthodontic treatment needs in prematurely born children[END_REF] analysed this same IOTN component (DHC) and obtained similar conclusions. Proven treatment need (grade 4 and 5) is also higher in premature patients compared to a control group. Souames et al. [START_REF] Souames | Orthodontic treatment need in French schoolchildren: an epidemiological study using the Index of Orthodontic Treatment Need[END_REF] evaluated the treatment need in French children using IOTN and indicated that 21.3% of children in the study had a proven orthodontic treatment need (grades 4 + 5). This value was compared to those obtained in other countries and it is therefore interesting to note that the treatment need varies by country. We find much higher percentage of grades 4 and 5 in our study for the non-premature group than Paulsson et al. [START_REF] Paulsson | Malocclusion traits and orthodontic treatment needs in prematurely born children[END_REF] and also much higher than that presented by Souames et al. [START_REF] Souames | Orthodontic treatment need in French schoolchildren: an epidemiological study using the Index of Orthodontic Treatment Need[END_REF] within the French population. This difference can be explained by the fact that the patients included in the control group of our study were patients treated in an orthodontic practice. They therefore had a higher treatment need than patients from the "general" population. Therefore, finding a significant difference between preterm and control groups is more difficult and we would certainly need to increase the power of the study with a larger number of patients. The higher treatment need in very and extremely preterm infants questions about the indication of a specific orthodontic treatment of these patients. Nevertheless, according to Cochrane, intellectual and motor management is still heterogeneous and still not agreed [START_REF] Spittle | Early developmental intervention programmes provided post hospital discharge to prevent motor and cognitive impairment in preterm infants. Cochrane Neonatal Group, éditeur[END_REF].

Conclusion

Premature babies suffer both from adverse foetal growth (in particular from the respiratory system immaturity) and from the iatrogenic effects of care performed immediately after delivery, such as the use of nasogastric probes. Some authors described orthodontic consequences, but the studies were contradictory. On the other hand, the differences, ethnic and about malocclusions management protocol according to the regions of the world, did not allow to extrapolate the results in France. In the end of this comparative epidemiological study on casts and x-rays of premature infants (47 children, 13 of which were extremely and very preterm) versus a control group of 150 non-premature children, we observed that: Preterm infants had significantly more bilateral posterior crossbites than term infants (p <0.05). Moderately preterm infants (born between 4 and 8 weeks before the term) were not significantly different from term-born ones. Extremely and very preterm babies (born more than 8 weeks before the term) had significantly more impacted teeth (54% versus 20%) and therefore a greater orthodontic treatment need than term-born children.

In the end of this study, we can conclude that if moderately preterm babies do not differ significantly from term-born infants, very preterm birth is a risk factor in orthodontics. Table I. Description of the study population (sex and age) for the preterm group (PT) and the control group (C).

Figures and Tables legends

Table II. Malocclusion traits distribution and comparison between the groups, extremely preterm and very preterm (EPT/VPT (A), N=13), moderately preterm (MPT (B), N=34), overall preterm (PT (A+B), N=47) and control (C, N=150).

Table III. Mean cephalometric values and comparison between the groups, extremely preterm and very preterm (EPT/VPT (A), N=13), moderately preterm (MPT (B), N=34), overall preterm (PT (A+B), N=47) and control (C, N=150).

Table IV.

Orthodontic treatment need and comparison between the groups, extremely preterm and very preterm (EPT/VPT (A), N=13), moderately preterm (MPT (B), N=34), overall preterm (PT (A+B), N=47) and control (C, N=150).

Figure 1 .

 1 Figure 1. Diagram showing the recruitment of preterm participants.

Table V .

 V IOTN grades percentage within the groups, extremely preterm and very preterm EPT/VPT (N=13), moderately preterm MPT (N=34), overall preterm PT (N=47) and control C (N=150).

TABLE I

 I

		PRETERM GROUP	CONTROL GROUP
			N=47		N=150	Group
		n	%	n	%	differences
	SEX
	Girls	88	58.67%	25	53.19%	NS
	Boys	62	41.33%	22	46.81%	NS
	Ratio G/B	1.42		1.14
		Mean	SD	Mean	SD
	AGE (years)	11.39	2.56	10.66	2.1	NS
	NS : Not Signifiant

Table II .

 II Malocclusion traits distribution and comparison between the groups, extremely preterm and very preterm (EPT/VPT (A), N=13), moderately preterm (MPT (B), N=34), overall preterm (PT (A+B), N=47) and control (C, N=150).

	Malocclusions					PT (A+B)
		n	%	n	%	n	%	n	%	A / C	B/C	A+B/C	A/B
	Class I	6	46.2%	18	52.9%	24	51.1%	88	58.7%	NS	NS	NS	NS
	Class II	6	46.2%	16	47.1%	22	46.8%	62	41.3%	NS	NS	NS	NS
	Class III	1	7.7%	0	0.0%	1	2.1%	0	0.0%	p<0,05	NS	NS	NS
	Deep-bite (> 4mm)	6	46.2%	18	52.9%	24	51.1%	51	34.0%	NS	NS	NS	NS
	Open-bite (> 0mm)	1	7.7%	2	5.9%	3	6.4%	5	3.3%	NS	NS	NS	NS
	Overjet (> 6mm)	2	15.4%	6	17.6%	8	17.0%	41	27.3%	NS	NS	NS	NS
	Overjet <0mm	1	7.7%	0	0.0%	1	2.1%	3	2.0%	NS	NS	NS	NS
	Anterior crossbite	2	15.4%	3	8.8%	5	10.6%	22	14.7%	NS	NS	NS	NS
	Post. unilat. crossbite	4	30.8%	10	29.4%	14	29.8%	33	22.0%	NS	NS	NS	NS
	Post. bilat. crossbite	3	23.1%	2	5.9%	5	10.6%	1	0.7%	p<0,05	NS	p<0,05	NS
	Total post. crossbite	7	53.8%	12	35.3%	19	40.4%	34	22.7%	NS	NS	NS	NS
	Crowding	4	30.8%	10	29.4%	14	29.8%	38	25.3%	NS	NS	NS	NS
	Diastema	1	7.7%	2	5.9%	3	6.4%	9	6.0%	NS	NS	NS	NS
	Rotations	8	61.5%	15	44.1%	23	48.9%	51	34.0%	NS	NS	NS	NS
	Additionnal teeth	0	0.0%	1	2.9%	1	2.1%	3	2.0%	NS	NS	NS	NS
	Agenesis	2	15.4%	3	8.8%	5	10.6%	15	10.0%	NS	NS	NS	NS
	Inclusions, retentions	7	53.8%	5	14.7%	12	25.5%	30	20.0%	p<0,05	NS	NS	NS
	Transpositions	1	7.7%	0	0.0%	1	2.1%	1	0.7%	NS	NS	NS	NS
	Anlyloses	0	0.0%	1	2.9%	1	2.1%	2	1.3%	NS	NS	NS	NS
	Values
		Mean SD	Mean SD	Mean SD	Mean SD	A / C	B/C	A+B/C	A/B
	Overjet (mm)	3.38	2.45	4.63	2.62	4.29	2.6	4.27	2.32	NS	NS	NS	NS
	Overbite (mm)	3.65	2.23	3.76	2.57	3.73	2.45	3.57	1.94	NS	NS	NS	NS

TABLE III

 III

	Values
		Mean SD	Mean SD	Mean SD	Mean SD	A / C	B/C	A+B/C	A/B
	Skeletal values
	SNA (°)	80.00	3.76	82.85	4.44	82.06 4.42	81.93	3.80	NS	NS	NS	NS
	SNB (°)	76.31	3.07	77.79	3.91	77.38 3.73	77.27	3.67	NS	NS	NS	NS
	ANB (°)	3.62	2.47	5.06	1.92	4.66	2.16	4.69	2.37	NS	NS	NS	NS
	FMA (°)	24.85	4.24	22.26	5.38	22.98 5.18	23.18	5.94	NS	NS	NS	NS
	Dental values
	Ao-Bo (mm)	0.54	3.11	0.57	2.64	0.56	2.76	0.21	2.96	NS	NS	NS	NS
	IMPA (°)	92.15	11.71	99.47	6.80	97.45 8.94	98.61	8.15	NS	NS	NS	NS
	I/F (°)	112.08 7.19	114.79 8.18	114.04 7.93	114.67 8.02	NS	NS	NS	NS

TABLE IV

 IV

				PRETERM GROUP			CONTROL GROUP
		EPT / VPT (A)	MPT (B)			(C)		Group Differences, P value
		EPT / VPT (A)	MPT (B)		PT (A+B)		(C )		Group Differences, P value
		EPT / VPT (A)	MPT (B)		PT (A+B)		(C)		Group Differences, P value
	IOTN grades	EPT / VPT (A)	MPT (B)		PT (A+B)		(C)		Group Differences, P value
		n	%	n	%	n	%	n	%	A / C	B/C	A+B/C	A/B
	Grade 1	0	0.00%	0	0.00%	0	0.00%	2	1.33%	NS	NS	NS	NS
	Grade 2	1	7.69%	8	22.86%	9	18.75%	45	30.00%	NS	NS	NS	NS
	Grade 1 + 2	1	7.69%	8	22.86%	9	18.75%	47	31.33%	NS	NS	NS	NS
	Grade 3	0	0.00%	9	25.71%	9	18.75%	25	16.67%	NS	NS	NS	NS
	Grade 1+2+3	1	7.69%	17	48.57%	18	37.50%	72	48.00%	NS	NS	NS	p<0,05
	Grade 4	4	30.77%	10	28.57%	14	29.17%	53	35.33%	NS	NS	NS	NS
	Grade 5	8	61.54%	8	22.86%	16	33.33%	25	16.67%	p<0,05	NS	NS	NS
	Grade 4 + 5	12	92.31%	18	51.43%	30	62.50%	78	52.00%	NS	NS	NS	NS
	Grade 3+4+5	12	92.31%	27	77.14%	39	81.25%	103	68.67%	NS	NS	NS	NS

TABLE V EPT

 V

		/ VPT (A)	MPT (B)	PT (A+B)	Control (C)
	Grades 1+2 (low treatment need)	7.70%	22.90%	18.80%	31.30%
	Grade 3 (moderate treatment need)	0%	25.70%	18.80%	16.70%
	Grades 4+5 (high treatment need)	92.30%	51.40%	62.50%	52%

Acknowledgments: We thank Mr. Nicolas Eydoux for reviewing this article. We also thank Dr. Marie-José Goumy and Dr. Claire Pernier, head of the Department of Orthodontics in the University Hospital of Lyon, who authorized the recruitment of patients in our study population.

Disclosure of interest: The authors declare that they have no competing interest.


Raul F Guerrero

Josep Valls-Fonayet

Tristan Richard
email: tristan.richard@u-bordeaux.fr

Emma Cantos-Villar

A rapid quantification of stilbene content in wine by ultra-high pressure liquid chromatography -mass spectrometry

Keywords: stilbene, viniferin, wine, mass spectrometry

Stilbenes are a family of bioactive phenolic compounds. Wine is one of the main sources of stilbenes in diet. Very few studies have dealt with a detailed quantitative analysis of stilbenes in wine. Most methodologies reported until now have been restricted to the analysis of few stilbenes such as resveratrol and piceid. In this study, a method for the quantification of wine stilbenes has been developed and validated. The method was simple, fast and sensitive with LOD between 4-28 μg/L. Matrix effects were assessed, and the methodology was validated in terms of precision, accuracy, linearity and repetitiveness. The method was able to quantify, in less than 5 minutes, fifteen targeted stilbenes in wines including seven monomers, three dimers, one trimer, and four tetramers. The methodology was applied to white and red wines. E-piceid was the main stilbene in white wine (mean 155 μg/L). In red wine, Z-and E-piceid (mean 3.73 and 3.16 mg/L, respectively) were predominant. Additionally, large amount of other stilbenes including oligomers such as hopeaphenol (mean 1.55 mg/L) were found in red wines. The developed methodology could be useful to reveal differences in the contents of stilbenes in wine depending on variety, season, terroir, treatments, among others and potentially be used as a quality wine marker.

Introduction

Wine is a complex evolving matrix in which a large number of compounds with different chemical nature coexist in a wide concentration ranges. In order to characterize such a complex mixture, the development of metabolomic approaches based on mass spectrometry has opened new opportunities to assess wine quality and traceability [START_REF] Alañón | Wine science in the metabolomics era[END_REF][START_REF] Arbulu | Untargeted metabolomic analysis using liquid chromatography quadrupole time-of-flight mass spectrometry for non-volatile profiling of wines[END_REF]. Concerning wine metabolomics, sometimes referred as Wineomics [START_REF] Wine-Omics | Wine-omics[END_REF], more than 2000 molecules have been described in wine, including primary wine metabolites such as sugars, amino acids, biogenic amines, organic acids, fatty acids or minerals, and secondary metabolites such as phenolics or volatile compounds [START_REF] Arbulu | Untargeted metabolomic analysis using liquid chromatography quadrupole time-of-flight mass spectrometry for non-volatile profiling of wines[END_REF].

Wine polyphenols constitute an heterogeneous family of chemical compounds belonging to several different chemical structures [START_REF] Quideau | Plant Polyphenols: Chemical Properties, Biological Activities, and Synthesis[END_REF].

All these phenolic compounds have attracted a enormous interest because of their organoleptic properties in wine that included aroma, colour, flavour, bitterness and astringency [START_REF] Garrido | Wine and grape polyphenols -A chemical perspective[END_REF]. In addition, their role as bioactive compounds have been widely reported as a key factor for the protection against cancer, cardiovascular, and neurodegenerative diseases [START_REF] Quideau | Plant Polyphenols: Chemical Properties, Biological Activities, and Synthesis[END_REF]. Because of their chemical complexity, the individual identification and quantification of all polyphenols remains a challenge (García-Guzmán, Hernández-Artiga, Palacios-Ponce de [START_REF] García-Guzmán | Selective methods for polyphenols and sulphur dioxide determination in wines[END_REF]. However, recent advances in liquid chromatography hyphenated with mass spectrometry have allowed a large improvement in the simultaneous detection and quantification of many polyphenols in wines in the last few years.

Around ninety anthocyanins and anthocyanin derivative pigments such as pyranoanthocyanins were characterized by liquid chromatography coupled triple quadrupole mass spectrometer (LC-QqQ-MS) in Sangiovese wines [START_REF] Arapitsas | Study of Sangiovese Wines Pigment Profile by UHPLC-MS/MS[END_REF]. Lambert et al developed also a method by LC-QqQ-MS for the selective quantification of up to 152 phenolic wine compounds, including 100 anthocyanins and derivatives, 15 phenolic acids, 5 flavonols, 11 flavanols and 6 stilbenes [START_REF] Lambert | A High-Throughput UHPLC-QqQ-MS Method for Polyphenol Profiling in Rosé Wines[END_REF].

Stilbenes are a particular interesting family of non-flavonoid polyphenols in wine, because of their health related properties [START_REF] Dvorakova | Anti-inflammatory activity of natural stilbenoids: A review[END_REF][START_REF] Temsamani | Piceatannol and other wine stilbenes: A pool of inhibitors against α-synuclein aggregation and cytotoxicity[END_REF][START_REF] Vang | What is new for an old molecule? systematic review and recommendations on the use of resveratrol[END_REF] and the fact that wine may represent the major source of these compounds in occidental diets, providing a up to 98% of their intake [START_REF] Zamora-Ros | Concentrations of resveratrol and derivatives in foods and estimation of dietary intake in a Spanish population: European Prospective Investigation into Cancer and Nutrition (EPIC)-Spain cohort[END_REF]. Recent studies also stated that stilbenes could play a role in the preservation of wine [START_REF] Raposo | Sulfur free red wines through the use of grapevine shoots: Impact on the wine quality[END_REF]. It opens the possiblity to identify the stilbene composition and concentration in wines as a quality marker.

Despite a large number of other phenolic compounds are indeed routinely analysed in wines, the stilbenes analysis is considerably reduced and the number of stilbenes analysed is often limited to the quantification of E-resveratrol, its glucoside E-piceid and their corresponding cis isomers.

Several reasons can be mentioned to explain this lack of convenient methodologies for the stilbene's analysis. Firstly, their contents in wines are usually quite low, in a range below of mg/L (ppm), which requires highly sensitive methods. Secondly, most of these minor compounds have been recently described. Finally, pure stilbene standards are not yet commercially available in order to allow a reliable quantification.

The development of new methodologies using LC-MS systems can provide a further improvement in the detection and quantification of wine stilbenes. In relation with identification methods, the stilbene profile by suspect screening analysis has recently been published. Flamini and co-authors identified eighteen potential stilbene derivatives to be present in two grape samples on the basis of accurate mass measurements and isotopic patterns by high resolution qTOF mass spectrometry [START_REF] Flamini | An innovative approach to grape metabolomics: stilbene profiling by suspect screening analysis[END_REF]. Also with a qTOF detector providing high resolution mass spectra, Moss et al. compiled 41 putative stilbene derivatives potentially present in red wine by screening precursor MS ions as well as characteristic neutral losses in the MS/MS fragments [START_REF] Moss | Investigation of monomeric and oligomeric wine stilbenoids in red wines by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry[END_REF]. The presence of some of these putative compounds was also confirmed thanks to the comparison with pure standards such as ε-viniferin, δ-viniferin, or pallidol. Although qTOF detectors can also be used for quantification purposes, both cited articles just focused on the qualitative analysis of grape and wine stilbenes. Because of their efficiency characteristics (sensitivity enhancement and time saving), triple quadrupoles working in multiple reaction monitoring (MRM) mode are the most often used mass spectrometers for quantification when standards are available, since they provide a large dynamic range, a great sensitivity and low LOD and LOQ [START_REF] Lambert | A High-Throughput UHPLC-QqQ-MS Method for Polyphenol Profiling in Rosé Wines[END_REF].

Despite the advances related to the detection of stilbenes in wine, the matter of their quantification by throughput methods remains relatively unexplored. Buiarelli et al. developed a 45-minute long LC-QqQ-MS methodology with a C18 column for the direct determination of resveratrol, piceid and astringin isomers. The method showed a good sensitivity and the detection limits were around 50 ng/mL [START_REF] Buiarelli | Analysis of some stilbenes in Italian wines by liquid chromatography/tandem mass spectrometry[END_REF]. Erhardt et al described the separation under ten minutes of 38 phenolic compounds in grapes, including 11 stilbenes piceatannol,isorhapontin,astringin;3 dimers: pallidol,a tetramer: isohopeaphenol), with LOD between 0.005 and 0.620 mg/kg grape [START_REF] Ehrhardt | Analysis of the phenolic composition of fungus-resistant grape varieties cultivated in Italy and Germany using UHPLC-MS/MS[END_REF]. Recently, Hurtado-Gaitan and co-authors have developed an LC-QqQ-MS method for the analysis of five grapevine stilbenes (4 monomers: resveratrol, piceid, piceatannol, pterostilbene; and a one dimer: ε-viniferin) in different complex matrices including red wine after solid phase extraction. The LOD were in the range of 0.04-0.12 mg/L (Hurtado-Gaitán et al. 2017).

In response to the lack of fast and simple methodologies to quantify stilbenes in wine, the aim of the current work was to develop and validate a fast LC-QqQ-MS methodology to quantify the fifteen main stilbenes described in grapevine. Among these compounds, six were quantified for the first time in wine. Finally, the method was validated and applied to real white and red wine samples.

Materials and methods

Stilbene standards and wine samples

E-astringin (Carbosynth, UK), E-piceid (Sigma Aldrich, Germany), E-piceatannol (ChromaDex, USA), E-resveratrol (Sigma Aldrich, USA), E-4-hydroxystilbene (Acros organics, Belgium) were commercially available. Ampelopsin A, hopeaphenol, isohopeaphenol, R2-viniferin, miyabenol C, ε-viniferin, R-viniferin, ω-Viniferin were isolated from a grapevine raw shoot following the method described by Biais and coauthors [START_REF] Biais | Antioxidant and cytoprotective activities of grapevine stilbenes. [Article][END_REF]. The cis isomers were obtained using UV-C irradiation (254 nm) from trans isomers [START_REF] Mattivi | Isolation, Characterization, and Evolution in Red Wine Vinification of Resveratrol Monomers[END_REF]. The white and red wines analyzed in this study were purchased in wine shops (Table 1S, in supplementary data).

Preparation of wine solutions

Ultrapure water from Milli Q Direct System (Merck Millipore, USA), L-(+)-tartaric acid (Merck, Germany), ethanol 96% v/v (pharma grade, Panreac, Spain), and sodium hydroxide pure pellets (pharma grade, Panreac, Spain) were used for wine matrix. For model wine solution (MW), tartaric acid (4 g) was diluted into 120 mL of ethanol 96% on 1 L volumetric flask. Solution was flushed with water up to 1 L. pH was adjusted with drops of sodium hydroxide 2 N solution up to 3.6. Standard white wine solution (WW) was composed of a mix of five monovarietal white wines made in the experimental winery IFAPA-Rancho de la Merced: Traminer, Vijiriega, Jaén blanco, Moscatel and Palomino fino. Into a Schott flask, 500 mL of each wine was poured to achieve 2.5 L of white wine matrix. Resulting solution showed 3.1 pH, 12.2% alc. vol., and 5.3 total acidity (g/L tartaric acid). Standard red wine solution (RW) was obtained as described for white using five monovarietal red wines: Pinot noir, Petit verdot, Malbec, Marselan and Tannat.

Resulted red wine matrix showed 3.6 pH, 13.9% alc. vol., and 5.1 total acidity (g/L tartaric acid).

Solutions were centrifuged during 20 min at 4000 rpm and filtrated through PTFE 0.45 μm filters.

Wine samples were diluted in a ratio 1:10 in MW solution. Subsequently, 20 µL of internal standard solution (E-4-hydroxystilbene) was added into 180 µL of sample to achieve a 1.28 ppm internal standard final concentration.

Instrumentation

Ultrapure water from Milli Q Direct System (Merck Millipore, USA), methanol for UHPLC (Merck, Germany) and formic acid 98-100% (Merck, Germany) were used. Compounds separation was performed on a Waters Acquity TQD LC/MS/MS System with photodiode array (PDA) detector equipped with a mass spectrometer Xevo TQD (Waters, USA). The column used was an Acquity UPLC BEH C18 (2.1 x 100 mm, 1.7 μm, Waters, USA). The mobile phases consisted of phase A: water 0.1% formic acid; and phase B: methanol 0.1% formic acid. The 6.60 min elution method at flow 0.35 mL/min was 0 min 10% B, 0.20 min 20% B, 1.60 min 40% B, 3.60 min 70% B, 4.20 min 100% B, 5.20 min 100% B, and recovering initial conditions, 5.60 min 10% B. Wash solvent was water/methanol in a ratio 50/50 and purge solvent was water 0.1% formic acid. Column temperature was kept at 40ºC and sample temperature at 10ºC. Injection volume was 10 μL for standards and samples. Mass spectrometer Xevo TQD was driven by software Masslynx v 4.1 (Waters) and set on electrospray negative ion mode (ES -).

Mass spectrometer was set on 2.30 kV capillary source voltage, 450ºC source desolvation temperature, 1000 L/h (N2) desolvation gas flow and 50 L/h cone gas (Argon) flow. Nitrogen generator from Peak Scientific (UK), and argon gas bottle (Air Liquide, France) were coupled to the mass detector for gas supplying. Dwell was automatically adjusted for minimum 12 points per peak and smoothing was applied on peaks. Smoothing method was on mean, 2 smooth iterations and 2 smooth widths.

Method development

Model wine (MW), standard white wine (WW) and standard red wine (RW) solutions were used to validate the method of stilbenes in wine analysis. Firstly, stilbene standards were separately dissolved in methanol/water in a ratio 50/50 to achieve an approximate concentration of 200 mg/L. Secondly, stilbene solutions were diluted in MW, WW and RW to achieve a concentration of 10 mg/L of each compound. These solutions were further diluted to achieve a second stock concentration of 5 mg/L. Finally, 5 mg/L solutions were dissolved in a ration 1/5 to achieve 1 mg/L solutions. These last solutions were used to evaluate the matrix effect. Solutions were prepared in triplicates. MW, WW, RW solutions and their dilutions were also injected with no standard addition.

The MW with 10 mg/L of each stilbene (MW-10ppm) solution was further used to prepare calibration curves. The E-4-hydroxystilbene was used as internal standard. This compound was firstly dissolved in methanol/water in a ratio 50/50 to achieve a 10 mg/L stock solution. The internal standard was added to each solution to achieve a 1 mg/L final concentration. Five serial dilutions were prepared from the MW-10ppm solution (5, 1, 0.5, 0.1 and 0.05 mg/L of each standard). Five serial dilutions were also prepared from 0.05 mg/L solution to achieve 0.02, 0.01, 0.005, 0.003 and 0.001 mg/L solutions. Calibration was prepared in duplicates and injected five times. Area value relation with internal standard area was used as quantification response.

Calibration was calculated considering origin forced inclusion and no weighting function.

Standards were injected to study linearity and accuracy (LOD and LOQ).

For intra-and interday effects, calibration curve with internal standard experiment was reinjected 5 days after. Vials were kept at 4ºC in a fridge. Relative standard deviation (RSD) at day 0 and day 5 were result of 5-times standard injection.

Commercial wine analysis

Wines were diluted in a ratio 1:10 in MW solution. Subsequently, 20 µL of internal standard solution (E-4-hydroxystilbene) was added into 180 µL of sample to achieve a 10 mg/L internal standard final concentration. All experiments were performed at least in triplicate. Data presented are means ± standard deviation.

Statistical analysis

Statistical analyses were performed using R scripts in BioStatFlow web application (biostatflow.org, v2.9).

Results and discussion

Analysis of individual stilbenes and selection of MRM conditions

Triple quadrupole mass spectrometers (QqQ-MS) are normally programmed in multiple reactions monitoring (MRM), where several transitions between the parent ion and their fragment ions are collected. The MRM mode used in LC-QqQ-MS methodology provides the selectivity required for the analysis by focusing on transitions that are specific to the quantified compounds [START_REF] Lambert | A High-Throughput UHPLC-QqQ-MS Method for Polyphenol Profiling in Rosé Wines[END_REF]. According to previous reports, the identification using MS/MS experiments would require the analysis of at least two product ions, the most intense one being used as a quantifier ion, while the other one is used as a qualifier (Kruve et al., 2015a(Kruve et al., , 2015b)).

In order to estimate the operational parameters concerning the optimal detection of the MRM, both the cone voltage and the collision energy were optimized by direct infusion. A total of fifteen stilbenes (Figure 1) were selected including seven monomers (E-and Z-astringin, E-and Z-piceid, piceatannol, E-and Z-resveratrol), three dimers (ampelopsin A, ε-and ω-viniferin), one trimer (miyabenol C), and four tetramers (hopeaphenol, isohopeaphenol, R2-and R-viniferin).

Each stilbene as well as the internal standard were infused in the detector and the MRM conditions were optimized in the negative mode. The two most intense fragment ions were selected to be used in the final LC-QqQ-MS method. All the optimization was performed using trans isomers, and then the transitions were checked to assure their suitability for the corresponding cis isomers. The selected parameters for each compound were those which had a better response. The results are reported in Table 1. A representative MRM chromatogram of a model wine spiked with 1 mg/L of each stilbene is presented in Figure 2. Under the described chromatographic conditions, stilbenes were analysed in less than six min.

The stilbene monomers fragmentation pattern was characterized by successive losses of 42 u fragments (ketene, CH2CO). So, for resveratrol, the precursor ion (m/z 227) gives two major ions: m/z 185 (elimination of one ketene molecule) and 143 (elimination of two ketene molecules). These ions correspond to previous reports [START_REF] Buiarelli | Analysis of some stilbenes in Italian wines by liquid chromatography/tandem mass spectrometry[END_REF], the ion at m/z 143 being the most abundant and in consequence it was selected as the quantifier ion. Similarly, for piceatannol (m/z 243) the fragments at m/z 201 and 159 represent again the loss of ketene molecules [START_REF] Wei | Stilbene profiles in different tissues of Vitis vinifera L. cv. Cabernet Sauvignon and a comparison of their antioxidant activity[END_REF]. The glucoside derivatives of these monomers also follow a similar pattern. The qualifier ions for piceid (m/z 389) and astringin (m/z 405) correspond to the loss of the glucose moiety (162 u), giving the fragment ions at m/z 227 and 243, respectively. This transition is generally the most commonly obtained for stilbene glucosides [START_REF] Buiarelli | Analysis of some stilbenes in Italian wines by liquid chromatography/tandem mass spectrometry[END_REF][START_REF] López-Hernández | Trans-stilbenes in commercial grape juices: quantification using HPLC approaches[END_REF].

Concerning dimers, ε-and ω-viniferin, share a parent ion at m/z 453 and a quantifier ion at m/z 359, which corresponds to the loss of the phenol ring (94 u) [START_REF] Ehrhardt | Analysis of the phenolic composition of fungus-resistant grape varieties cultivated in Italy and Germany using UHPLC-MS/MS[END_REF]. The ωviniferin gives also a fragment at m/z 347, consistent with a C7H6O loss (106 u), in accordance with previous results [START_REF] Ehrhardt | Analysis of the phenolic composition of fungus-resistant grape varieties cultivated in Italy and Germany using UHPLC-MS/MS[END_REF][START_REF] Moss | Investigation of monomeric and oligomeric wine stilbenoids in red wines by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry[END_REF]. Regarding ampelopsin A (m/z 469), it gives two major ions at m/z 451 (loss of H2O) and 363 (loss of 106 u, 4methylenecyclohexan-2,5-dienone). The trimer miyabenol C (m/z 679) quantifier signal at m/z 345 has been previously reported [START_REF] Vrhovsek | Profiling and accurate quantification of trans-resveratrol, trans-piceid, transpterostilbene and 11 viniferins induced by Plasmopara viticola in partially resistant grapevine leaves[END_REF]. It would correspond to the loss of two molecules of 4-methylenecyclohexan-2,5-dienone (106 u), a CO group (28 u), and a phenol (94 u). The corresponding qualifier ion at m/z 451 is formed after the loss of a 4methylenecyclohexan-2,5-dienone (106 u), a phenol (94 u) and a CO group [START_REF] Moss | Investigation of monomeric and oligomeric wine stilbenoids in red wines by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry[END_REF].

Four tetramers (ion precursor at m/z 905) were included in the method. The fragment at m/z 359 was observed for all of them, and is the main transition for hopeaphenol and isohopeaphenol.

This ion has been suggested to arise from the symmetrical splitting of the tetramer molecule and the additional loss of a phenol [START_REF] Moss | Investigation of monomeric and oligomeric wine stilbenoids in red wines by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry[END_REF]. In addition, hopeaphenol and isohopeaphenol share the main qualifier ion at m/z 451 -which would correspond to the loss of a dimer [START_REF] Moss | Investigation of monomeric and oligomeric wine stilbenoids in red wines by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry[END_REF]. Meanwhile, R2-viniferin has as a quantifier ion at m/z 811, consistent with the loss of one phenol group (94 u). The R-viniferin gives a fragment at m/z 799, which consistent to the loss of a 4-methylencyclohexan-2,5-dienone (106 u).

Method validation and quality parameters

The validation of the LC-QqQ-MS method for quantification of the fifteen selected stilbenes was performed by investigating the following quality parameters: linearity, limits of detection (LOD) and quantification (LOQ), repeatability (intra-and inter-day), and recovery in each wine matrixes (MW, WW and RW) (Kruve et al., 2015a(Kruve et al., , 2015b)). First at all, the possible matrix effects were investigated. These effects result from co-eluting matrix compounds that compete for ionization capacity inducing a decrease or increase of the analyte signal [START_REF] Choi | Effect of liquid chromatography separation of complex matrices on liquid chromatography-tandem mass spectrometry signal suppression[END_REF]. Standard white (WW) and red wine (RW) solutions containing 1 mg/L of each stilbene were analyzed.

The same experience was repeated with different dilutions of WW and RW solutions. For the undiluted wines, the results clearly showed a matrix effect for almost all compounds with a mean recovery rate of 50 ± 30% for the RW solution (Table 2S, supplementary data). The effect is especially significant for the most polar compounds in the RW solution. The astringin recovery rate drops to 16% in the undiluted RW solution. Diluting the samples in the model wine solution increases the recovery rate. In order to reduce matrix effects, a dilution in 1/10 ratio appears to be the most appropriate for quantifying stilbenes with the minimal ionization suppression. The mean recovery rates increased to 101 ± 9% and 79 ± 10% in WW and RW solutions, respectively.

Due to the matrix effects an internal standard was added in the method (Kruve et al., 2015b). An ideal internal standard should mimic closely the properties of the analyte, differ only slightly chemically, and have desirable chromatographic properties such as stable isotopes [START_REF] Wieling | LC-MS-MS experiences with internal standards[END_REF]. Such ideal internal standard for the stilbenes analysis is right now unachievable for practical reasons: first, most stilbenes are simply not commercially available in their natural form, and secondly the few isotopes available would represent a too high added expensiveness for the analysis. A compromise had to be found and among available stilbene compounds hydroxystilbene was selected. The main reason was its analogy with the stilbenes selected, its absence in wine, and its availability as a commercial standard.

Quality parameters of the LC-QqQ-MS method were reported in the Tables 2 and3. The linearity range of the method was evaluated by serial dilution of a stock solution of the studied compounds in the model wine solution (range 0.001 to 10 mg/L of each stilbene). The correlation coefficients (R 2 ) were ranged between 0.981 and 0.999 depending on the analytes.

The regression equations and the linearity ranges of each stilbene are reported in Table 2. One of the main characteristics of the MRM methodology is the large dynamic range analyte quantification from few μg/L to mg/L in our case. The LOD and LOQ values were calculated using the classical signal-to-noise ratio criterion of 3 and 10, respectively. The values for the selected stilbenes are given in Table 2. LOQ values were ranged between 15 and 61 μg/L. These results are coherent with those obtained by other QqQ methodologies [START_REF] Buiarelli | Analysis of some stilbenes in Italian wines by liquid chromatography/tandem mass spectrometry[END_REF]Hurtado-Gaitán et al., 2017). For example, Hurtado-Gaitan et al. reported LOQ of 220, 70, 150 and 90 μg/L for resveratrol, piceid, piceatannol and ε-viniferin, respectively, but with a lower injection volume (Hurtado-Gaitán et al., 2017).

Recovery of each stilbene was calculated in MW, WW and RW solutions containing 1 mg/L of each stilbene. The data obtained were presented in Table 3. For MW and WW solutions, recovery values for the stilbenes were within the range of 82 and 120% with a mean recovery rate of 105 ± 12% and 101 ± 9%, respectively. In contrast, in the RW solution the recovery values were lower in the range of 69 and 108% with a mean recovery rate of 79 ± 10%. This decrease of recovery rate in red wines could be due to matrix effects as previously observed.

Recovery values higher than 100% are not unusual in LC-MS and have been previously reported for stilbenes analysis (Rodríguez-Cabo et al., 2014). Low recovery values for several analytes due to matrix effects in wine have also been previously observed. For example, Lambert et al.

found intensity losses of over 50% in QqQ analysis of phenolic acids when the concentration of formic acid is not optimized [START_REF] Lambert | A High-Throughput UHPLC-QqQ-MS Method for Polyphenol Profiling in Rosé Wines[END_REF].

Same experiments were conducted with wine solutions containing 0.5 mg/L of each stilbene. The obtained recovery rates were similar 97 ± 12%, 93 ± 12%, and 72 ± 10% for MW, WW and RW solutions, respectively. Finally, concerning the reproducibility, the relative standard deviation (RSD%) in term of concentration, was determined in each wine matrix (MW, WW and RW solution). The results are given in Table 3. The RSD% values were around 10% except for miyabenol C and R-viniferin.

Evaluation of stilbene pattern in wines

The optimized LC-QqQ-MS method was applied to determine the content of stilbenes in different commercial mono-varietal white and red wines (Table 1S, supplementary data).

Different cultivars and vintages were selected to observe the wide spectra application of the method. Stilbene concentration in grape, and therefore in wine, is affected by climate, type of soil, year, variety, winemaking processes, among others, and therefore wine stilbene concentration hugely varies [START_REF] Bavaresco | Effects of elicitors, viticultural factors, and enological practices on resveratrol and stilbenes in Grapevine and Wine[END_REF].

Analysis of white wines

Ten commercial Spanish white wines were analysed from eight different varieties: Albariño (×3), Chardonnay, Godello, Moscatel, Riesling, Sauvignon blanc, Verdejo, and Viura. The content of stilbenes in these white wines is summarized in Table 4.

The total stilbene content in white wines was ranged between 0.04 and 0.56 mg/L with a mean value of 0.23 mg/L. These values are in agreement with literature data (Lamuela-Raventós, Romero-Pérez, Waterhouse, & de la [START_REF] Lamuela-Raventós | Direct HPLC Analysis of cis-and trans-Resveratrol and Piceid Isomers in Spanish Red Vitis vinifera Wines[END_REF][START_REF] De Lima | Determination of stilbenes (trans-astringin, cis-and transpiceid, and cis-and trans-resveratrol) in Portuguese wines[END_REF]. A total of twelve stilbenes were identified in white wines. But only six compounds were identified above the limits of quantification (E-and Z-astringin, E-and Z-piceid, E-and Z-resveratrol). The E-piceid (0.11-0.33 mg/L, mean 0.16 mg/L) was the most dominant stilbene in all white wines followed by a pool of three compounds its isomer Z-piceid, and the two isomers of astringin (11-44 μg/L, mean 21 μg/L). In agreement with others works, piceid seems to be the main stilbene in white wines [START_REF] De Lima | Determination of stilbenes (trans-astringin, cis-and transpiceid, and cis-and trans-resveratrol) in Portuguese wines[END_REF]. The concentrations in astringin and resveratrol were significantly lower than previously reported in white wines [START_REF] Lamuela-Raventós | Direct HPLC Analysis of cis-and trans-Resveratrol and Piceid Isomers in Spanish Red Vitis vinifera Wines[END_REF][START_REF] De Lima | Determination of stilbenes (trans-astringin, cis-and transpiceid, and cis-and trans-resveratrol) in Portuguese wines[END_REF], which may be explain due to the huge number of factor that influence the stilbene concentration. Finally, the presence of stilbene oligomers was observed for the first time in some white wines including one dimer (ε-viniferin), one trimer (miyabenol C), and four tetramers (hopehaphenol, isohopeaphenol, R-and R2-viniferin). However, it was not possible to quantify them because their concentrations were under the limit of quantification.

Analysis of red wines

Ten commercial Spanish red wines were investigated from seven different varieties: Cencibel, Garnacha, Merlot (×2), Monastrell, Syrah, Tempranillo (×3), and Tintilla de Rota. The content of stilbenes in these red wines is reported in Table 5.

In red wines, total stilbene concentration is much more variable than in white wines. Depending on the wine, stilbene content was ranged from 0.40 mg/L to 35.5 mg/L (mean 13.1 mg/L). The stilbenes encountered in red wines are mostly glucosylated (Table 3S). Depending of the red wine, between 40 and 100% (mean 68%) of the stilbene quantified were glucosides. In agreement with previously described for white wines, piceid (sum E-and Z-isomers) was the main stilbene in red wines (Total 0.28-15.7 mg/L, mean 6.89 mg/L). The two isomer levelsare comparable (mean 3.16 and 3.73 mg/L for E-and Z-piceid, respectively). This result is in agreement with literature data even if their concentrations in some red wines were slightly higher than those previously reported [START_REF] Moreno-Labanda | Determination of piceid and resveratrol in Spanish wines deriving from Monastrell (Vitis vinifera L.) grape variety[END_REF]. In contrast with white wines, the second main stilbene in red wines is resveratrol (Total nd-9.84 mg/L, mean 3.19 mg/L). As piceid, the resveratrol concentrations in red wines are strongly contrasted. Levels of E-and Z-resveratrol are similar (mean 1.38 and 1.81 mg/L in E-and Z-resveratrol, respectively). The astringin isomers are quantified in all red wines. Levels of E-astringin (0.06-2.99 mg/L, mean 0.67 mg/L) are significantly higher than that of Z-astringin (0.06-0.23 mg/L, mean 0.10 mg/L).

The monomer piceatannol is only quantified in three wines. Even if the results are contrasted, red wines may contain relative high amount of stilbene oligomers up to 11.2 mg/L representing 13% of the total stilbene content (Table 3S). The tetramers are the most representative oligomeric stilbenes. The isohopeaphenol is the main oligomer quantified (nq-7.47 mg/L, mean 1.55 mg/L), while its isomer hopeaphenol is only quantified in two wines. In addition, R2-viniferin is detected for the first time in wine. Red wine R10 (Monastrell, Table 1S) is the only wine which showed quantifiable amount of miyabenol C, ε-and ω-viniferin (1.41, 0.81 and 0.31 mg/L, respectively). Monastrell grape has been described as high resveratrol producer [START_REF] Gatto | Ripening and genotype control stilbene accumulation in healthy grapes[END_REF], being Monastrell wine reported as a high resveratrol content wine [START_REF] Moreno-Labanda | Determination of piceid and resveratrol in Spanish wines deriving from Monastrell (Vitis vinifera L.) grape variety[END_REF].

As far as we know, it is the first time that quantitative analysis has been reported for Z-astringin, hopeaphenol, isohopeaphenol, R2-viniferin, miyabenol C and ω-viniferin, in wine. Quantitative data on stilbene monomers E-and Z-resveratrol, E-and Z-piceid has usually been reported [START_REF] Guerrero | Phenolic characterisation of red grapes autochthonous to Andalusia[END_REF][START_REF] Lamuela-Raventós | Direct HPLC Analysis of cis-and trans-Resveratrol and Piceid Isomers in Spanish Red Vitis vinifera Wines[END_REF][START_REF] Romero-Pérez | Levels of cis-and trans-resveratrol and their glucosides in white and rosé Vitis vinifera wines from spain[END_REF]. Vitrac et al., reported also data on astringin, δ-viniferin and ε-viniferin in Brazilian wines [START_REF] Vitrac | Determination of stilbenes (δ-viniferin, trans-astringin, trans-piceid, cis-and trans-resveratrol, ε-viniferin) in Brazilian wines[END_REF]. Moss et al., were able to detect piceatannol, astringin, ε-viniferin, ω-viniferin, miyabenol C and hopeaphenol in a red wine extract (100-folds concentrated) but not to quantify them by direct injection ultra-highperformance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry [START_REF] Moss | Investigation of monomeric and oligomeric wine stilbenoids in red wines by ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry[END_REF].

Principal component analysis (PCA) was used to compare white and red wines (Figure 3). The percentage of total variability explained by PC1 was about 76%. A separation between white and red whites was observed. The comparison of the scores plot and the loadings plot showed a tendency to have higher concentration in stilbenes in red wines. In fact, it is widely known that stilbene concentration in white wines is lower than in red wines because in red winemaking the must, grape skin and often seeds are in contact during the alcoholic fermentation process [START_REF] Fernandez-Marin | Isorhapontigenin: A novel bioactive stilbene from wine grapes[END_REF]. The PCA highlights the great dispersion in stilbene content between red wines.

Conclusion

Resveratrol shows a large range of biological effects, including cancer, cardioprotective, neuroprotective preventions. During the past decades, other natural derivatives of resveratrol were identified in plant kingdom and more specifically in grapevine. These compounds received particular attention for their beneficial effects but their content in wine remains relatively unstudied. The developed method enables the identification and quantification of fifteen stilbenes well known in wine. The method is fast, does not require sample preparation and presents a large dynamic range between few μg/L to few mg/L. In addition, this method may permit increase the number of quantifiable stilbenes as new compounds might be identified.

Concerning their content in wine, twelve stilbenes were quantified in red wines. These wines present a wide dispersion of stilbenes that could be due to numerous factors such as grape variety, biotic and abiotic stresses or winemaking processes. In further studies, research on the impact of these and other factors to control the stilbene content especially in red wine may be affordable due to the development on the described method.

HO OH R 1 R 2 R 1 R 2 E-resveratrol OH H E-piceid Oglc H piceatannol OH OH E-astringin Oglc OH OH OH R 1 R 2 R 1 R 2 Z-resveratrol OH H Z-piceid Oglc H Z-

Figure 1 .

 1 Figure 1. Stilbene structures.

Figure 2 .

 2 Figure 2.Representative MRM chromatograms of a model wine spiked with 1 mg/L of each stilbene. The transition of quantification is shown for each compound.

Figure 3 .

 3 Figure 3. PCA score and loading plots of the two principal components of white (open circles) and red wines (full squares).

Table 1

 1 Compound name, retention time, and optimized MRM conditions for the analyses of the studied stilbenes by UPLC-MS/MS.

		Qualifier
	Compound	Rt (min) Parent Ion (m/z)

Table 2 .

 2 Linearity data, limit of detection (LOD), and limit of quantification (LOQ) of the stilbenes. Z-isomers not included in the table were quantified with the calibration curve obtained for the E-isomers.

	Compound	Calibration equation	Correlation coefficient (R 2 )	Linearity (mg/L)	LOD (mg/L)	LOQ (mg/L)
	E-astringin	y = 5.75x + 0.212	0.9959	0.03 -3.0	0.009	0.030
	ampelopsin A	y = 4.07x -0.160	0.9985	0.01 -5.0	0.005	0.015
	hopeaphenol	y = 1.08x + 0.051	0,9837	0.02 -3.0	0.007	0.021
	E-piceid	y = 8.43x + 0.260	0.9843	0.01 -7.5	0.004	0.012
	isohopeaphenol	y = 2.38x + 0.087	0.9811	0.03 -7.5	0.011	0.033
	E-piceatannol	y = 1.87x + 0.084	0.9947	0.06 -4.0	0.018	0.061
	E-resveratrol	y = 3.20x + 0.180	0.9775	0.03 -4.0	0.010	0.030
	R2-viniferin	y = 0.36x -0.113	0.9870	0.06 -3.0	0.020	0.060
	miyabenol C	y = 0.14x -0 .015	0.9992	0.08 -5.0	0.028	0.084
	ε-viniferin	y = 0.32x + 0.030	0.9936	0.05 -5.0	0.017	0.051
	R-viniferin	y = 0.99x -0.262	0.9940	0.03 -10.0	0.010	0.030
	ω-viniferin	y = 1.07x -0.060	0.9990	0.03 -10.0	0.010	0.030

Table 3 .

 3 Average recovery, intra-and inter-day precision of the concentration of the stilbenes of standard solution (1 mg/L of each compound) in the different wine matrixes (model wine solution, white wine, and red wine).

		Model wine			White wine			Red wine
	Compound	Average	Intraday	Interday	Average	Intraday	Interday	Average	Intraday	Interday
		recovery (%)	(RSD %)	(RSD %)	recovery (%)	(RSD %)	(RSD %)	recovery (%)	(RSD %)	(RSD %)
	astringin	109	11	7	101	4	7	75	3
	ampelopsin A	103	10	8	94	9	6	69	6
	hopeaphenol	107	9	5	105	11	6	80	10
	piceid	107	9	7	99	8	7	72	6
	isohopeaphenol	102	9	6	98	10	6	72	8
	piceatannol	110	9	11	120	12	7	79	7
	resveratrol	108	11	4	101	11	2	71	6
	R2-viniferin	82	5	15	116	12	13	108	8
	miyabenol C	116	15	12	97	14	11	87	21
	ε-viniferin	130	10	8	99	8	6	74	13
	R-viniferin	83	24	10	103	19	10	79	14
	ω-viniferin	107	8	6	88	11	6	82

Table 4 .

 4 Content of stilbenes (in μg/L) in commercial white wines. The figure in brackets represents the standard deviation.

		White wines
	Compound
		W1	W2	W3	W4	W5	W6	W7	W8	W9	W10	Mean
	E-astringin	15 (1)	19 (3)	19 (1)	11 (3)	18 (3)	16 (3)	32 (3)	24 (4)	16 (4)	44 (2)	22 (9)
	ampelopsin A	nq	nq	nq	nq	nq	nq	nq	nq	nq	nq	-
	hopeaphenol	nd	nq	nd	nd	nd	nd	nd	nd	nd	nq	-
	E-piceid	nq	110 (6)	129 (8)	180 (14) 150 (10) 160 (7)	150 (6)	120 (8)	220 (45) 330 (21) 155 (84)
	isohopeaphenol nd	nd	nd	nd	nd	nd	nd	nd	nd	nq	-
	piceatannol	nq	nq	nq	nq	nq	nq	nq	nq	nq	nq	-
	Z-astringin	21 (5)	9 (2)	11 (3)	12 (15)	15 (7)	11 (2)	16 (3)	48 (2)	14 (3)	20 (4)	18 (11)
	E-resveratrol	nd	nq	nq	nq	nq	nq	nq	nq	19 (13)	49 (10)	7 (16)
	R2-viniferin	nd	nq	nd	nd	nq	nd	nq	nq	nq	nd	-
	Z-piceid	8 (6)	14 (3)	11 (2)	18 (4)	17 (3)	22 (3)	23 (3)	49 (17)	29 (20)	42 (9)	23 (13)
	miyabenol C	nq	nd	nq	nq	nd	nq	nq	nd	nq	nq	-
	ε-viniferin	nq	nq	nq	nd	nq	nq	nq	nq	nq	nq	-
	Z-resveratrol	nd	nd	nq	nq	nq	nq	nq	nq	18 (14)	73 (22)	9 (23)
	R-viniferin	nq	nd	nd	nq	nq	nq	nq	nq	nq	nq	-
	ω-viniferin	nq	nq	nq	nq	nq	nq	nq	nq	nq	nq	-
	Total	44 (12)	152 (14) 170 (14) 229 (36) 200 (23) 209 (15) 221 (15) 241 (21) 316 (95) 558 (67) 234 (134)

Table 5 .

 5 Content of stilbenes (in μg/L) in commercial red wines. The figure in brackets represents the standard deviation.

		Red wines
	Compound
		R1	R2	R3	R4	R5	R6	R7	R8	R9	R10	Mean
	E-astringin	57 (6)	233 (18)	443 (29)	212 (12)	340 (13)	530 (30)	540 (23)	549 (29)	825 (110)	2999 (179)	673 (846)
	ampelopsin A	nq	nq	nq	nq	nq	nq	nq	nq	nq	nq	-
	hopeaphenol	nq	nq	nq	nq	nq	nq	nq	311 (32)	nq	494 (21)	81 (175)
	E-piceid	152 (13)	794 (85)	1020 (66)	1070 (70)	1780 (106) 2940 (207) 6570 (530)	3360 (250)	6820 (1060)	7118 (547)	3162 (2714)
	isohopeaphenol nq	490 (41)	353 (22)	nq	2020 (106) 1290 (62)	1150 (65)	2696 (169)	nq	7469 (597)	1547 (2714)
	piceatannol	nq	nq	nq	nq	nq	nq	nq	740 (48)	370 (55)	271 (19)	138 (251)
	Z-astringin	64 (10)	74 (9)	63 (6)	142 (13)	50 (4)	83 (3)	102 (4)	145 (7)	84 (7)	234 (12)	104 (56)
	E-resveratrol	nd	278 (52)	209 (14)	332 (27)	714 (62)	808 (92)	2656 (268)	3755 (38)	3577 (65)	1503 (160)	1383 (1433)
	R2-viniferin	nq	nq	nq	nq	nq	nq	nq	930 (39)	nq	3280 (109)	421 (1046)
	Z-piceid	125 (12)	999 (109)	1207 (72)	1462 (92)	2024 (142) 2800 (210) 6908 (542)	5457 (399)	8871 (1330)	7469 (597)	3732 (3149)
	miyabenol C	nq	nq	nq	nq	nq	nq	nq	nq	nq	1411 (116)	141 (446)
	ε-viniferin	nq	nq	nq	nq	nq	nq	nq	nq	nq	805 (47)	81 (255)
	Z-resveratrol	nq	302 (52)	nq	232 (17)	537 (46)	1025 (86)	2213 (233)	6085 (538)	3792 (67)	2129 (271)	1813 (2023)
	R-viniferin	nq	nq	nq	nq	nd	nq	nq	nq	nq	nq	-
	ω-viniferin	nq	nq	nq	nq	nq	nq	nq	nq	nq	305 (12)	31 (96)
	Total	399 (41)	3170 (366) 3295 (209) 3450 (231) 7465 (479) 9476 (690) 20139 (1665) 24028 (1665) 24339 (2687) 35487 (2666) 13125 (11975)

Acknowledgments

The authors gratefully thank Actichem (Montauban, France) for providing grapevine products and to Waters support (MA Cortés and J. Rodriguez) for their contribution in the development of the method. The work was supported by the Bordeaux Metabolome Facility and MetaboHUB (ANR-11-INBS-0010 project) and by INIA (RTA2015-00005-C01).

Funding

Notes

The authors declare no competing financial interest.