M.D Tuula Outinen
email: tuula.outinen@uta.fi

M.D Satu M Mäkelä

M.D Ilpo O Ala-Houhala

Heini Huhtala

M.D Mikko Hurme

Daniel H Libraty

M.D Simo S Oja

M.D Ilkka H Pörsti

M.D Jaana T Syrjänen

M.D Antti Vaheri

Tuula ; Outinen

Jukka ; Mustonen

M.D Tuula K Outinen

M.Sc S A Huhtala

M.D Jukka T Mustonen

High activity of indoleamine 2,3-dioxygenase is associated with renal insufficiency in Puumala hantavirus induced nephropathia epidemica

Keywords: Puumala hantavirus, nephropathia epidemica, kynurenine, tryptophan, indoleamine 2, 3-dioxygenase Puumala hantavirus, nephropathia epidemica, kynurenine, tryptophan, indoleamine 2, 3-20 dioxygenase

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Introduction 1 2 Nephropathia epidemica (NE) is a mild type of hemorrhagic fever with renal syndrome (HFRS), 3 caused by Puumala virus (PUUV) [START_REF] Vapalahti | Hantavirus 13 infections in Europe[END_REF]]. The virus is a member of the Hantavirus 4 genus in the 5

Bunyaviridae family and is carried by bank voles (Myodes glareolus) [START_REF] Vapalahti | Hantavirus 13 infections in Europe[END_REF]. NE is 6 prevalent in Finland, elsewhere in Scandinavia, in the Balkan region, and also in many parts of 7

Western Europe [START_REF] Kanerva | Pathogenesis of Puumala and other hantavirus infections[END_REF][START_REF] Vapalahti | Hantavirus 13 infections in Europe[END_REF]. Approximately 1000-3000 8 serological diagnoses of PUUV infection are made in Finland annually (http://www3.ktl.fi/), and 9 the seroprevalence in the population is 5% [Brummer- [START_REF] Brummer-Korvenkontio | 14 Epidemiological study of nephropathia epidemica in Finland 1989-96[END_REF]. Other 10 hantaviruses causing HFRS include Hantaan, Dobrava, Saaremaa, and Seoul viruses; while in the 11 Americas, Sin Nombre, Andes, and Black Creek Canal viruses cause hantavirus cardiopulmonary 12 syndrome (HCPS) [START_REF] Vapalahti | Hantavirus 13 infections in Europe[END_REF]. [START_REF] Brummer-Korvenkontio | 14 Epidemiological study of nephropathia epidemica in Finland 1989-96[END_REF] The clinical severity of NE varies greatly and host genetics have been shown to influence the 14 clinical picture [START_REF] Mustonen | Genetic 19 susceptibility to severe course of nephropathia epidemica caused by Puumala hantavirus[END_REF][START_REF] Mäkelä | Human leukocyte antigen-B8-DR3 is a more important risk factor for severe Puumala 23 hantavirus infection than the tumor necrosis factor-alpha(-308) G/A polymorphism[END_REF]. The usual symptoms are high fever, 15 headache, backache, abdominal pain and nausea, while hemorrhagic manifestations are uncommon 16 [START_REF] Lähdevirta | Nephropathia epidemica in Finland. A clinical histological and epidemiological 22 study[END_REF]Mustonen et al., 1994a;[START_REF] Settergren | Clinical aspects of nephropathia epidemica (Puumala virus infection) in Europe: 1 a review[END_REF]. Renal involvement is manifested by 17 proteinuria, hematuria, and oliguria followed by polyuria. A minority of patients require transient 18 dialysis treatment [START_REF] Lähdevirta | Nephropathia epidemica in Finland. A clinical histological and epidemiological 22 study[END_REF]Mustonen et al., 1994a;[START_REF] Settergren | Clinical aspects of nephropathia epidemica (Puumala virus infection) in Europe: 1 a review[END_REF]. The characteristic 19 histopathologic renal finding is acute tubulointerstitial nephritis [Mustonen et al., 1994b]. Complete 20 recovery is the usual outcome [START_REF] Lähdevirta | Nephropathia epidemica in Finland. A clinical histological and epidemiological 22 study[END_REF]Mustonen et al., 1994a;[START_REF] Settergren | Clinical aspects of nephropathia epidemica (Puumala virus infection) in Europe: 1 a review[END_REF]. Typical 21 laboratory findings in NE are leukocytosis, thrombocytopenia, anemia, and elevation of plasma C-22 reactive protein (CRP) and creatinine concentrations [Mustonen et al., 1994a;[START_REF] Settergren | Clinical aspects of nephropathia epidemica (Puumala virus infection) in Europe: 1 a review[END_REF]. In 23 addition radiological pulmonary manifestations have been detected in 16-53% of hospitalized 24 [START_REF] Kanerva | Pulmonary 9 involvement in nephropathia epidemica: radiological findings and their clinical correlations[END_REF][START_REF] Linderholm | Pulmonary involvement in 20 nephropathia epidemica as demonstrated by computed tomography[END_REF][START_REF] Lähdevirta | Nephropathia epidemica in Finland. A clinical histological and epidemiological 22 study[END_REF]Mustonen et al., 1 1994a;[START_REF] Paakkala | Medical imaging in 1 nephropathia epidemica and their clinical correlations[END_REF]. 2 Indoleamine 2,3-dioxygenase (IDO) is an enzyme catalyzing the first and rate-limiting step in the 3 major pathway of tryptophan (trp) catabolism to kynurenine (kyn) and its derivatives [Mellor, 4 2005]. IDO is expressed widely in various immune cells, including monocyte-derived macrophages 5 and dendritic cells [START_REF] Mellor | IDO expression by dendritic cells: tolerance and tryptophan 3 catabolism[END_REF]. It is also expressed in other types of cells, such as 6 certain tumour-cells, fibroblasts, and renal tubular epithelial cells (TEC) [START_REF] Burke | The role of indoleamine 2,3-dioxygenase in the 17 anti-tumour activity of human interferon-gamma in vivo[END_REF]Jalili et 7 al., 2007;[START_REF] Mohib | Proapoptotic activity of indoleamine 2,3-5 dioxygenase expressed in renal tubular epithelial cells[END_REF]. Interferon (IFN)-γ is the strongest inducer of IDO [Mellor and Munn, 8 2004]. Increased IDO activity results in the depletion of trp and this can lead to inhibition of T-cell 9 responses and proliferation and thus to immunosuppression [Hwu et al., 2000;Mellor et al., 2002]. 10 Depletion of trp is also thought to be the mechanism by which IDO activity inhibits the replication 11 of various bacteria, intracellular parasites, and viruses [START_REF] Takikawa | Biochemical and medical aspects of the indoleamine 2,3-dioxygenase-initiated 3 L-tryptophan metabolism[END_REF]. 12 In the present study, we investigated whether serum IDO concentrations are associated with 13 disease severity and renal insufficiency in Puumala hantavirus-induced HFRS. The rate of trp degradation reflects the IDO enzyme activity, and IDO level can thus be measured 5 by determining the ratio of kynurenine to tryptophan (kyn/trp) in sera [START_REF] Schrocksnadel | Monitoring tryptophan metabolism in 20 chronic immune activation[END_REF]. 6

Serum trp and kyn concentrations were measured afterwards from frozen samples by reverse-phase 7 high-performance liquid chromatography (HPLC), as previously described [START_REF] Laich | More rapid method for simultaneous 15 measurement of tryptophan and kynurenine by HPLC[END_REF]. Trp 8 was separated with a Shimadzu liquid chromatograph LC-10AD VP (Shimadzu Co, Kyoto, Japan) 9 using a 50-mm BDS Hypersil C18 5 µm column (Thermo Electron Co, Bellefonte, PA, USA). In order to describe the data, medians and ranges were given for continuous variables and 20 numbers and percentages for categorical variables. To evaluate the associations of plasma IDO 21 levels with the severity of NE, we divided the patients into two groups according to the maximum 22 IDO value. Groups were compared using the Mann-Whitney U-test. Categorical data were 23 analyzed by the x 2 test or the Fisher's exact test. Correlations were calculated by means of 24 Spearman's rank correlation coefficient. Wilcoxon's test was used to compare two related samples. 25 We also performed a logistic regression analysis with significant renal insufficiency (creatinine 1 >250 µmol/l) as a dependent factor and high IDO and low urinary output as independent factors in 2 order to further examine the associations of these factors with significant renal insufficiency. Age 3 and sex were also included in these models. Odds ratios (OR) and their 95% confidence intervals 4 (95% CI) were given. All tests were two-sided, and statistically significant P-values are given. All 5 analyses were made with the SPSS (version 7.5) statistical software package. 6 7 8

Results

10

The clinical and laboratory characteristics of the NE patients are shown in Table 1. The median 11 duration of symptoms before admission to the hospital was four days (range 1-15 days). None of the 12 patients was in clinical shock at the time of admission. Five of the 102 NE patients (5%) eventually 13 needed dialysis treatment during hospital stay. The median duration of fever was six days (range 2-14 19 days). Everyone recovered completely. [START_REF] Mustonen | Renal biopsy findings and clinicopathologic correlations in nephropathia epidemica[END_REF] The ability of maximum serum kyn/trp ratio to predict serum creatinine levels >250 µmol/l was 16 evaluated using receiver operating characteristics (ROC) curves [START_REF] Boyd | Mathematical tools for demonstrating the clinical usefulness of biochemical 9 markers[END_REF]. A maximum kyn/trp 17 >202 µmol/mmol showed a sensitivity of 85% and a specificity of 75% for detecting maximum 18 serum creatinine levels >250 µmol/l and the AUC (area under curve) was 0.84 (95% CI 0.76-0.91) 19

(Figure 1). This cut-off point was used to divide patients into two groups. Patients with low IDO 20 value had maximum kyn/trp ratio ≤202 µmol/mmol and patients with high IDO value had 21 maximum kyn/trp ratio >202 µmol/mmol. IDO values during acute illness and hospitalization were 22 significantly higher (median maximum kyn/trp 199.3, range 46.6-3679.2, µmol/mmol) than 23 convalescent values taken 14-32 days (median 22 days) after the onset of fever (median 64.7, range 24 23.9-350.6, µmol/mmol, P<0.001). 25 The proportion of males and females did not differ between patients with high serum IDO levels 1 and patients with low IDO levels (70% male vs. 65% male, high vs. low IDO groups, respectively, 2 P=0.618). Forty-six (45%) of the patients were smokers. The proportion of smokers also did not 3 differ between high and low IDO groups (52% smokers vs. 44% smokers, high vs. low IDO groups, 4 P=0.404). All five patients, who needed dialysis treatment were in the high IDO group (P=0.025). 5

The maximum level of IDO was associated with several other parameters reflecting disease severity 6 and renal impairment (Table 2). Patients with high IDO levels were older than patients with low 7 IDO levels, while BMI did not differ between these two groups. There was a strong positive 8 correlation between maximum serum IDO and creatinine levels (r=0.672, P<0.001), as well as 9 maximum IDO level and change in weight, which reflects fluid retention during renal impairment 10 (r=0.526, P<0.001). Maximum IDO levels and minimum urinary output were inversely correlated 11 (r=-0.385, P<0.001). There was a positive correlation between maximum IDO levels and the length 12 of hospital stay (r=0.494, P<0.001). Maximum IDO levels and the maximum blood leukocyte count 13 were positively correlated (r=0.508, P<0.001). We also performed a logistic regression analysis in 14 order to evaluate the association of high IDO levels with significant renal insufficiency 15 (creatinine>250 µmol/l) when adjusted for age, sex, and low urinary output (Table 3). A high IDO 16 level was an independent risk factor for significant renal insufficiency in this model. 17 (r=-0.201, P=0.044), the maximum blood leukocyte value (r=-0.244, P=0.014) and maximum CRP 1 (r=-0.256, P=0.009). 2

As the patients with NE sought medical assistance at different time intervals from the onset of 3 fever, the serum trp and kyn samples were also taken at different periods from the onset. From the 4 majority of patients (80 %) we had trp and kyn samples taken six days from the onset of fever. The 5 median kyn/trp level on that day was 191.9, range 40.9-2442.5 µmol/mmol. In this subgroup of 82 6 patients, the main results remained the same. Patients with high IDO (kyn/trp >202 µmol/mmol) six 7 days after the onset had higher maximum blood leukocytes (median 12.3, range 6.3-31.2 x10 9 /l vs. 8 median 9.1, range 5.1-24.0 x10 9 /l, P=0.001), higher maximum serum creatinine (median 445, range 9 79-1285 µmol/l vs. median 113 range 60-615 µmol/l, P<0.001), greater change in weight (median 10 3.5, range 0-12.0 kg vs. median 1.6, range 0-9.9 kg, P<0.001), and longer hospital stay (median 7, 11 range 3-14 days vs. median 5, range 2-10 days, P<0.001) than patients with low IDO. They also had 12 lower minimum urinary output (median 1085, range 50-4900 ml vs. median 1555, range 200-4940 13 ml, P=0.042) than patients with low IDO. 14 In order to analyze the kinetics of the changes in serum IDO and creatinine levels, we depicted 15 their daily medians in relation to the day of the onset of fever. Figure 2 shows that both variables 16 first gradually increased to their peak values, and thereafter they started to decline. Median IDO 17 peaked to 243.9 µmol/mmol nine days after the onset of fever, whereas median creatinine was at its 18 highest (265 µmol/l) 10 days after the onset of fever. 19 University Hospital using the same laboratory as in the present study. In SLE patients the median 4 IDO levels were 39.1-43.0 µmol/mmol during different seasons [START_REF] Pertovaara | 3 Indoleamine 2,3-dioxygenase activity is increased in patients with systemic lupus 4 erythematosus and predicts disease activation in the sunny season[END_REF]. In the 5 same study the median kyn/trp ratio was also analyzed in 309 healthy controls and was found to be 6 25.9 µmol/mmol. Interestingly, in bacteremic patients, the median maximum kyn/trp ratio was 7 lower (89.9 µmol/mmol) than in our patients with Puumala virus infection (199.3 µmol/mmol) 8 [START_REF] Huttunen | High activity of indoleamine 2,3 dioxygenase enzyme predicts disease 23 severity and case fatality in bacteremic patients[END_REF]. In the present study, the IDO levels were lower during the convalescent 9 phase (14-32 days after the onset of fever) than during the acute phase, but still higher than the 10 values published earlier for the healthy controls. It was not possible to standardize dietary intake of 11 the essential amino acid trp in our study. However, it seems unlikely that dietary intake would have 12 affected serum kyn/trp levels, since kyn/trp ratio is not affected by the dietary intake of trp 13 [START_REF] Schrocksnadel | Monitoring tryptophan metabolism in 20 chronic immune activation[END_REF]. 14

In the present study, high IDO levels were associated with increased disease severity, especially 15 renal impairment in acute Puumala hantavirus infection. A high IDO level was found to be an 16 independent risk factor for maximum serum creatinine exceeding 250 µmol/l, when examined 17 together with low urinary output, age, and sex. We found that high IDO levels were associated with 18 significant renal insufficiency and higher blood leukocyte count as well as with lower urinary 19 output and blood hematocrit. It also associated strongly with the duration of hospitalization. 20 IDO has not been widely studied in patients with viral infections. Increased IDO activity has been 21

found in acute dengue virus infection and chronic hepatitis C virus infection [START_REF] Becerra | Increased activity of indoleamine 2,3-dioxygenase in serum from acutely infected 4 dengue patients linked to gamma interferon antiviral function[END_REF]22 Larrea et al., 2007]. Also increased trp degradation has been found in chronic Epstein-Barr virus 23 infection [START_REF] Bellmann-Weiler | IFN-gamma 6 mediated pathways in patients with fatigue and chronic active Epstein Barr virus-infection[END_REF]. In those studies the association between IDO level and the 24 severity or progression of the disease was not studied. However, in human immunodeficiency virus 25 complications, e.g. weight loss and neuropsychiatric disorders [START_REF] Schroecksnadel | 22 Indoleamine-2, 3-dioxygenase and other interferon-gamma-mediated pathways in patients 23 with human immunodeficiency virus infection[END_REF]. High 2 IDO levels were also associated with severe disease and mortality in bacteremic patients [Huttunen 3 et al., 2010]. Treatment with granulocyte-macrophage colony-stimulating factor resulted in reduced 4 IDO levels in severe sepsis, possibly due to improved antibacterial defence [START_REF] Schefold | Treatment with granulocyte-16 macrophage colony-stimulating factor is associated with reduced indoleamine 2,3-17 dioxygenase activity and kynurenine pathway catabolites in patients with severe sepsis and 18 septic shock[END_REF]. 5

In the present study, a high IDO level was associated with increased severity of Puumala hantavirus 6 infection. 7

Studies of IDO in humans with kidney diseases are also scarce. It has been demonstrated that 8 concentrations of trp metabolites increase in chronic renal insufficiency [START_REF] Saito | Mechanism of increases in L-kynurenine and quinolinic acid in renal insufficiency. 8[END_REF]Schefold 9 et al., 2009]. This is presumed due to an increase in production and/or decrease in degradation, 10 rather than a decrease in renal excretion [START_REF] Saito | Mechanism of increases in L-kynurenine and quinolinic acid in renal insufficiency. 8[END_REF]. In our study, the level of trp metabolite, 11 kyn, positively correlated with the severity of acute renal insufficiency. This may suggest that the 12 increased production of kyn reflecting the activity of IDO is important. In kidney allograft 13 recipients, upregulation of IDO is associated with rejection [START_REF] Brandacher | Non-invasive monitoring of kidney 12 allograft rejection through IDO metabolism evaluation[END_REF]. In mice, it has 14 been shown that IDO promotes renal ischemia-reperfusion injury [START_REF] Mohib | Indoleamine 2,3-8 dioxygenase expression promotes renal ischemia-reperfusion injury[END_REF]. On the other 15 hand, in nephrotoxic serum nephritis, a model of crescentic glomerulonephritis, IDO acts as a 16

protective factor [START_REF] Hou | Inhibition of indoleamine 2, 3-dioxygenase-mediated 19 tryptophan catabolism accelerates crescentic glomerulonephritis[END_REF]. 17

The pathogenesis of NE is not well understood. PUUV has no direct cytopathic effect on cultured 18 cells [START_REF] Temonen | 8 Susceptibility of human cells to Puumala virus infection[END_REF].Therefore, it is unlikely that direct viral cytotoxicity is the primary 19 cause of pathology in NE. PUUV causes acute tubulointerstitial nephritis and the severity of 20 histological changes in NE has been found to be related to the clinical severity of renal failure as 21 measured by the highest serum creatinine level [Mustonen et al., 1994b]. In kidney biopsies of 22 patients with NE, there is an increased amount of infiltrating cells in the peritubular areas [Temonen 23 et al., 1996]. The infiltrating cells include plasma cells, monocytes/macrophages, and lymphocytes, 24 as well as polymorphonuclear cells, mainly eosinophils and neutrophils. CD8+ T-cells predominate 25 inhibition of IDO augments TEC survival [START_REF] Mohib | Proapoptotic activity of indoleamine 2,3-5 dioxygenase expressed in renal tubular epithelial cells[END_REF]. We found that high IDO was 7 strongly associated with significant renal failure. We also found that IDO levels peaked before 8 creatinine levels, which might suggest that IDO could be in fact involved in the pathogenesis of 9 renal failure in NE. One pathogenetic mechanism could be TEC apoptosis. Previously signs of 10 epithelial cell apoptosis have been detected in patients with PUUV infection [Klingström et al., 11 2006]. Increased IDO levels could also contribute to immunosuppression through T-cell 12 suppression and apoptosis. [START_REF] Brummer-Korvenkontio | 14 Epidemiological study of nephropathia epidemica in Finland 1989-96[END_REF] In conclusion, a high serum kyn/trp level reflecting increased IDO activity was associated with 14 clinically severe PUUV-induced interstitial nephritis and we suggest that it might even be involved IDO=kynurenine/tryptophan ratio. OR=odds ratio. CI=confidence interval. 5

  It 10 was monitored by fluorescence with a Shimadzu RF-10A XL detector at 266 nm excitation and 366 11 nm emission wavelengths. Kyn was separated with a Hewlett Packard 1100 liquid chromatograph 12 (Palo Alto, CA, USA) using a Merck LiChroCart 55-4150 mm cartridge containing a Purospher 13 STAR RP-18 3 µm column (Merck Co, Darmstadt, Germany). It was determined by ultraviolet 14 absorption at 360-nm wavelength with a Hewlett Packard G13144 detector. The kyn/trp ratio was 15 calculated by relating concentrations of kyn to trp, this allowing estimation of IDO activity.

  We also analysed maximum kyn and trp values separately in relation to parameters reflecting the 18 severity of NE. Maximum kyn level were positively correlated with maximum serum creatinine 19 (r=0.785, P<0.001) and change in weight (r=0.517, P<0.001). It was inversely correlated with 20 minimum urinary output (r=-0.357, P<0.001). There was also a positive correlation between 21 maximum kyn level and the length of hospital stay (r=0.517, P<0.001) and the maximum blood 22 leukocyte value (r=0.516, P<0.001). Maximum trp level had a slight positive correlation with 23 minimum urinary output (r=0.230, P=0.026), and a slight inverse correlation with change in weight 24

  study with 102 consecutive and prospectively identified hospitalized patients is to our 24 knowledge the first study on IDO during a hantavirus infection. Our data show that IDO levels are 25 Puumala hantavirus infection. The median maximum kyn/trp ratio in our 1 study was 199.3 µmol/mmol during acute NE. Previous studies on IDO levels in patients with 2 systemic lupus erythematosus (SLE) and in bacteremic patients have been carried out in Tampere 3

  degradation by IDO was associated with disease progression and 1

  infiltrate[START_REF] Temonen | Cytokines, 5 adhesion molecules, and cellular infiltration in nephropathia epidemica kidneys: an 6 immunohistochemical study[END_REF]. This is thought to indicate that cell-mediated 1 cytotoxicity is important in the kidney involvement in NE. 2 Macrophages express IDO when exposed to IFN-γ. Increased IDO activity in turn results in T-cell 3 supression and the induction of T-regulatory cells (T-regs). By a positive feedback loop, T-regs can 4 further enhance IDO activity by stimulating increased IFN-γ expression [Mulley and Nikolic-5 Paterson, 2008]. It has been demonstrated that increased IDO activity promotes TEC apoptosis and 6

Figure 1 . 5 6Figure 2 .

 152 Figure 1. Receiver operating characteristics (ROC) curve for maximum kynurenine/tryptophan ratio 4

14 15 16 Materials and Methods 17

	1	inflammatory bowel disease, diabetes mellitus, psychiatric disorder, migraine and hyperplasia of the
	2	prostate in two patients; sick sinus syndrome treated with pacemaker, osteoporosis, psoriasis,
	3 3	Sjögren's syndrome, mitral valve disease, epilepsy, fibromyalgia, sarcoidosis, multiple sclerosis, Basel, Switzerland). Blood cell count was completed by hematological cell counters by Bayer.
	4 4	polyneuropathia, chronic gastritis, transient ischaemic attack, congenital hearing impairment, Serum creatinine was determined by Cobas Integra analyzer.
	5	operated atrial septal defect, and operated melanoma in one patient each.
	6	The diagnosis of acute PUUV infection was serologically confirmed in all cases [Vapalahti et al.,
	7	1996]. All subjects gave informed consent before participation and the study was approved by the
	8 9 10	F o r Ethics Committee of Tampere University Hospital and the University of Massachusetts Medical School. F o r
	11	Study protocol	P P
	12		e e
	13	e r All 102 patients were studied during the acute phase of NE. A detailed past and current medical e r
	14 15	R history was obtained, and a careful physical examination was performed. Blood samples to analyze serum trp and kyn, creatinine, CRP, and blood cell count were collected for up to five consecutive R
	18 19 16 17 18 19	Patients days after hospitalization. Other blood samples were taken according to the clinical needs of the e v i e w e patient. The highest and the lowest value of each patient of the various variables measured during hospitalization were designated as the maximum and minimum values. In this study, we have v i e defined significant renal insufficiency as a serum creatinine value exceeding 250 µmol/l. Minimum w
	20 20	The study cohort consisted of 102 prospectively identified consecutive patients with acute NE urinary output was defined as low if it was equal or lower than the median value in the study
	21 21	treated at Tampere University Hospital, Finland, between September 2000 and January 2008. The population (≤1440 ml/day) and high if it was >1440 ml/day.
	22 22	median patient age was 46 (range 22-77) years, and 69 (68%) were males.
	23 23	Thirty-eight (37%) patients had one or more of the following diseases before NE: essential Methods
	24 24	hypertension in 12, dyslipidemia in eight, arthritis rheumatoides and atrial fibrillation/fluctuation in
	25	four, and coronary artery disease and bronchial asthma in three patients; hypothyreosis, chronic

Table 1 .

 1 Clinical and laboratory findings in 102 patients with acute nephropathia epidemica.

	15
	16	in the pathogenesis of renal failure in NE.
	17
	18
	20
	21	The study was financially supported by the Competitive Research Funding of the Pirkanmaa
	22	Hospital District, the European Commission Project "Diagnosis and control of rodent-borne viral
	23	zoonoses in Europe" (QLK2-CT-2002-01358) and the U.S. National Institutes of Health (NIH grant
	24	U19 AI57319).

Page 19 of 23 John Wiley & Sons Journal of Medical Virology

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	20

Table 2 .

 2 Clinical and laboratory findings in 102 patients with nephropathia epidemica divided into 1 two groups according to maximum serum kynurenine/tryptophan ratio.

	2
	3
		IDO max ≤202	IDO max >202	P
		µmol/mmol	µmol/mmol
		n=52	n=50
	Age (years)	38 (22-77)	50 (25-74)	0.002

Table 3 .

 3 Multivariate analysis of risk factors for serum creatinine level >250 µmol/l among 94 1

	2	hospitalized patients with nephropathia epidemica.
	3
				Creatinine ≤250	Creatinine >250	OR	95% CI
				µmol/l	µmol/l
				N=53	N=41
		Age (years)		43	48	0.98	0.94-1.03
		Sex	Female	18	9	1	reference
			Male	35	32	2.70	0.80-9.09
		Low urinary output No	33	14	1	reference
			Yes	20	27	1.96	0.65-5.87
		High IDO	No	39	6	1	reference
			Yes	14	35	17.57	5.25-58.77
	4	Continuous variables are expressed as medians. Category variables are expressed as numbers.

Acknowledgements 19


BSc (Hons) PhD Jeff S Millership
email: j.millership@qub.ac.uk

Cchem Frsc

Microassay of Drugs and Modern Measurement Techniques

Keywords: paediatric, drug analysis, pharmacokinetics < Drugs, pharmacodynamics < Drugs paediatric, drug analysis, pharmacokinetics, pharmacodynamics

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Within the neonatal and paediatric population it is well known that unlicensed and off-label drug use is widespread [START_REF] Turner | Unlicensed and off label drug use in paediatric wards: prospective study[END_REF][START_REF] Conroy | Unlicensed and off label analgesic use in paediatric pain management[END_REF][START_REF] Conroy | Unlicensed and off-label drug use in neonates[END_REF][START_REF] Mcintyre | Unlicensed and off-label prescribing of drugs in general practice[END_REF][START_REF] Chalumeau | Off label and unlicensed drug use among French office based paediatricians[END_REF][START_REF] Jong | Unlicensed and off label prescription of drugs to children: population based cohort study[END_REF]. There have been numerous attempts to overcome these difficulties with such proposals as the FDA modernisation act of 1997 [START_REF]Food and Drug Administration Moderization Act of[END_REF], the Best Pharmaceuticals for Children Act 2002 [START_REF]Best Pharmaceuticals for Children Act[END_REF] and the Pediatric Research Equity Act 2003 [START_REF][END_REF].

Within the European Union the EU Regulation on Paediatric Medicines (10) was adopted in 2006 and came into force in 2007. These proposals are aimed not only at providing licensing information for older, off-patent drugs but also ensuring that drugs being brought to the market are licensed for children, if appropriate.

In order for licensing data and age-appropriate dosing guidelines to be drawn up for this population it is necessary for pharmacokinetic/pharmacodynamic studies to be undertaken. In pharmacokinetic studies the drug level concentrations are determined in biological matrices and it is this aspect that will form the basis of this review, with a focus on the trend to develop micro-analytical methods suitable for studies in children and neonates.

Determination of drugs in biological matrices

In the study of pharmacokinetics/pharmacodynamics established procedures involve the determination of the drug/metabolites in plasma/serum samples. In early papers in this area it was often the case that relatively large volumes of serum/plasma were used in the analytical methodology. One paper by Roure et al [START_REF] Roure | Pharmacokinetics of alfentanil in children undergoing surgery[END_REF] details a study of the pharmacokinetics of alfentanil in children undergoing surgery. The methodology describes the collection of twelve 2 ml blood samples over a five-hour period for use in the radioimmunoassay, which appears to require 1 mL of plasma although it should be pointed out that the limit of detection was 0.1 ng/mL. Similarly, in the early days of high performance liquid chromatography (HPLC), due to the sensitivity of the detection systems and the need to determine the low [clinical] concentrations of the drug during the elimination phase in pharmacokinetic studies, relatively large volumes of biological matrices were required. For example Cooper et al [START_REF] Cooper | High pressure liquid chromatographic determination of hydrochlorothiazide in human serum and urine[END_REF] reported a method for the determination of hydrochlorothiazide in serum and urine. The determination utilises 1 mL of serum and involves liquid-liquid extraction, the chromatographic method involved reverse phase HPLC with UV detection (271 nm) and the method was linear in the range 200-800 ng/mL. These authors comment, even in 1976, on the volume of serum used and indicate that that in their method the volume of serum can be reduced to less than 200 µL for studies in children, thus suggesting the need for micro-analytical methods for such studies in children even at that time. It is worth noting that a recent article [START_REF] Gao | Simultaneous quantitation of hydrochlorothiazide and metoprolol in human plasma by liquid chromatography-tandem mass spectrometry[END_REF] described the determination of hydrochlorothiazide (and metopropolol) by an LC/MS/MS requiring only 100 µL of plasma with a linear of the range 3 -1000 ng/mL thus showing the changes in methodology and sensitivity over 35 years.

It is now generally accepted that in developing assays for pharmacokinetic studies in children there is a necessity to utilise sampling techniques that require as small a sample volume as possible. In order to review the developments of modern micro-analytical methods the following sections will focus on chromatographic methods since in recent history these are the mostly widely used techniques with the following aspects detailed:- Indometacin is a drug used in neonates for the induction of closure of patent ductus arteriosus and because of its narrow therapeutic window monitoring is recommended. Thomas et al [START_REF] Thomas | Quantitation of indomethacin in serum and plasma using gas chromatography-mass spectrometry (GC-MS)[END_REF] have detailed the determination of this drug via LLE from serum or plasma followed by determination using GC/MS/MS. Either 200 µL of serum or plasma is treated with the internal standard (IS) solution, buffer and extracted with dichloromethane. The organic layer is separated and evaporated to dryness and the residue silylated. The silylated samples were then analysed by GC/MS using selected ion monitoring (SIM) with linearity over the range 0.25 -10 mg/mL. Similarly Dailly et al [START_REF] Dailly | Population pharmacokinetics of domperidone in preterm neonates[END_REF] have reported the use of LLE in a HPLC determination of domperidone in plasma in a pharmacokinetic study involving pre-term neonates. In this method 400 µL of plasma was used in the extraction and analysis involved reverse phase HPLC and florescence detection resulting in a LOQ of 2 ng/mL although the linear range was not detailed.

Al-Asmari et al [START_REF] Al-Asmari | Method for the quantification of diamorphine and its metabolites in pediatric plasma samples by liquid chromatographytandem mass spectrometry[END_REF] have reported a method for the determination of diamorphine (DIM) and metabolites in plasma; the method being applied in a study of the use of intranasal administration of diamorphine in children with acute to severe pain. The method involved SPE with < 250 µL plasma, which was devised so as to minimise hydrolysis of DIM during the procedure and HPLC/MS/MS. The method used was capable of determining diamorphine and metabolites and used gradient elution and detection involving electrospray ionisation in the positive ion mode with MRM detection. The paper is worthy of study for details of the extensive validation including a study of matrix effects. Skolnik et al [START_REF] Skolnik | A liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of actinomycin-D and vincristine in children with cancer[END_REF] have developed a HPLC/MS/MS method involving SPE for the simultaneous determination of both vincristine and actinomycin D [which are used in the treatment of various paediatric cancers] in paediatric plasma samples. The method utilises 500 µL of plasma which was mixed with IS standard solution and then subjected to SPE. The LC/MS/MS involved reverse phase chromatography and detection using ESI (positive ion mode) and MRM. The method is reported to be linear, for both compounds over the range 0.5 -100ng/mL with 0.5ng/mL being the LLOQ. Similarly Herd et al [START_REF] Herd | Ketamine disposition in children presenting for procedural sedation and analgesia in a children's emergency department[END_REF] have reported a LC/MS/MS method for the determination of ketamine and norketamine in children utilising SPE. In this methodology a 200 µL blood sample was mixed with 50 µL of IS solution containing deuterated ketamine and norketamine and 3 mL of phosphate buffer. The solution was sonicated for 15 min, centrifuged and the subjected to SPE. The reverse phase HPLC incorporated a MS system comprising a Turbolon spray source used in the positive ion mode with MRM. The method was linear for ketamine over the range 0.125 -4.5 mg/mL and 0.06 -2.0 mg/mL for norketamine.

Salm et al (19) have detailed a LC/MS/MS for the determination of tacrolimus and cyclosporin in whole blood. These workers comment on the problems associated with conventional immunoassay for these two compounds due to cross reactivity with metabolites and difficulties with low hematocrit on the determination of tacrolimus. PP was carried out by the addition of a zinc sulphate solution to a 100 µL blood sample, followed by the addition of a solution containing two IS (ascomycin and cyclosporine D), this mixture was simply mixed, centrifuged and the supernatant injected onto the LC/MS/MS system. The reverse phase HPLC incorporated APCI used in the negative ionisation mode. The method was linear for tacrolimus over the range 1-30 µg/L and 2-2000 µg/L for cyclosporin.

Muller et al [START_REF] Muller | Pharmacokinetics of penicillin G in infants with a gestational age of less than 32 weeks[END_REF] have investigated the pharmacokinetics of penicillin G in infants with a gestational age of less than 32 weeks using a simple HPLC method with UV detection (215 nm). The method utilises only 100 µL of serum and the sample prep is straightforward involving addition of IS solution (methicillin in methanol), vortexing for 30 s, cooling at -20 o C for 10 min, vortexing again for 30 s, centrifugation and filtration before injection of the supernatant. Similarly Metsvaht et al ( 21) have described a pharmacokinetic study of penicillin G in very low birth weight neonates. The analytical method is similar to that detailed above [HPLC/UV detection at 200 nm] but uses only 50 µl of plasma, which is treated with acetonitrile, vortexed and centrifuged and then the supernatant is injected onto the HPLC system.

In GC it is often the case that derivatisation techniques are necessary (e.g. in the indometacin assay above) in order to produce satisfactory chromatography, however, in HPLC derivatisation is rarely required to optimise the chromatography because of the larger number of parameters that can be modified in HPLC compared to GC. In some cases, however, researchers have reported the HPLC analysis of drugs using derivatisation methods.

Topiramate (Figure 1), an anticonvulsant drug, has no chromophore and thus is inactive in terms of UV/visible or fluorescence detection. Vovk et al [START_REF] Vovk | A nonlinear mixed effects modelling analysis of topiramate pharmacokinetics in patients with epilepsy[END_REF] have detailed a method for the determination of topiramate in plasma that is applicable to pharmacokinetic studies in both adults and children with epilepsy. This method involves derivatisation using 9fluorenylmethyl chloroformate (Fig 1; 2), which reacts with the amide function of topiramate to give a fluorescent label so that the compound can be determined with high sensitivity. The method (based on the work of Bahrami et al ( 23)) involves a simple extraction of topiramate from 250 µL of plasma with dichloromethane, the organic extract was evaporated to dryness

Dried Blood Spot sampling

History

Dr Robert Guthrie was an American scientist who had a particular interest in the genetic disorder phenylketonuria (PKU). With PKU there is a lack of the enzyme phenylalanine hydrolase, which results in a build up of phenylalanine in the body and this can lead to a number of serious problems including mental retardation. Guthrie [START_REF] Guthrie | A Simple Phenylalanine Method For Detecting Phenylketonuria In Large Populations Of Newborn Infants[END_REF] was searching for a simple method of blood sampling in neonates so that the levels of phenylalanine could be determined to check for PKU and therefore dealt with to prevent the known problems. This led to the development of the "Guthrie Card" as detailed in Figure 2.

Use for drug assay

Nowadays these cards are used to collect heel prick blood samples from neonates during their first few days of life, the samples are then sent to a central laboratory where they can be screened for a whole series of inborn errors of metabolism including PKU, congenital hypothyroidism, cystic fibrosis and sickle cell disorders. Following their use in the investigation of inborn errors of metabolism other workers sought to utilise this sample collection method to determine drug concentrations in whole blood samples. The first example reported in 1978 was the use of DBS for the determination of theophylline [START_REF] Albani | A simple rapid gas chromatographic method for the determination of theophylline in dried whole blood on filter paper cards[END_REF].

Later an alternative EMIT method for the determination of theophylline in DBS [START_REF] Rattenbury | Measurement of theophylline in dried blood spots by spectrophotometric enzyme immunoassay[END_REF] was reported and this was applied to the study theophylline levels in asthmatic children and the acceptance of domicilary DBS collection [START_REF] Rattenbury | Acceptance of domiciliary theophylline monitoring using dried blood spots[END_REF]. During the 1980/90s further, intermittent applications were reported detailing the determinations of for example chloroquine (28), mefloquine ( 29) sisomicin [START_REF] Fujimoto | Fluorometric determination of sisomicin, an aminoglycoside antibiotic, in dried blood spots on filter paper by reversed-phase highperformance liquid chromatography with pre-column derivatization[END_REF], gentamicin and netilmicin [START_REF] Fujimoto | Fluorescence polarization immunoassay of gentamicin or netilmicin in blood spotted on filter paper[END_REF], quinine, hydroxychloroquine, chloroquine, and desethylchloroquine [START_REF] Croes | Simple and rapid HPLC of quinine, hydroxychloroquine, chloroquine, and desethylchloroquine in serum, whole blood, and filter paper-adsorbed dry blood[END_REF], dapsone, monoacetyldapsone, and pyrimethamine [START_REF] Rønn | High-performance liquid chromatography determination of dapsone, monoacetyldapsone, and pyrimethamine in filter paper blood spots[END_REF].

Over the last ten years applications specifically related to the use of DBS sampling in clinical areas, therapeutic drug monitoring (TDM) and more specifically paediatric studies began to appear [START_REF] Watson | A rapid and sensitive method for the determination of the amount of theophylline in blood spots[END_REF][START_REF] Oliveira | A simple microanalytical technique for the determination of paracetamol and its main metabolites in blood spots[END_REF][START_REF] Millership | Drug level determination in the paediatric population using blood spots: applications in unlicensed and off-label drug use[END_REF]. In one of these articles Oliveira et al [START_REF] Oliveira | A simple microanalytical technique for the determination of paracetamol and its main metabolites in blood spots[END_REF] reported the determination of paracetamol and its main metabolites in dried blood spots, the developed method being applied to the analysis of DBS samples obtained from neonates. The method involved reverse phase HPLC with UV detection and was capable of determining paracetamol (PA), paracetamol glucuronide (PAG) and paracetamol sulphate (PAS). The DBS were extracted using a formate buffer solution, the IS was then added followed by acetonitrile for protein precipitation, this solution was then centrifuged and the supernatant evaporated to dryness before reconstitution in formate buffer. The analytical range for the 3 compounds was reported to be 40-2000 ng/mL for PA and 160-4000 ng/mL for PAG and PAS. The authors report the application of the methodology in neonates by presenting the PA concentration time profile for a neonate following a rectal dose of PA.

Damen et al (37) have recently reported an updated HPLC method for the determination of vincristine and actinomycin-D in dried blood spots. In this method the preparation of standard DBS samples involved the spiking of whole blood (collected from healthy volunteers) and the application of 40 µL volumes onto DBS cards. From these samples circles 0.25 inches in diameter were cut, extracted with acetonitrile/methanol/water (1:1:1, v/v/v) containing IS for 15 min with sonification. Following the extraction the sample was simply centrifuged and the clear supernatant injected onto the LC/MS/MS, which employed electrospray ionisation detection operating in the positive ion mode and MRM. The assay for vincristine was linear from 1 to 100 ng/mL and actinomycin-D from 2 to 250 ng/mL and was tested on samples obtained by a finger prick and the authors indicating the applicability of the method to routine clinical investigations. Although this methodology was not specifically developed for paediatric studies it is worth comparing the method with that above for a similar determination of these same two drugs in plasma by Skolnik et al [START_REF] Skolnik | A liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of actinomycin-D and vincristine in children with cancer[END_REF]. In the plasma method 500 mL was used which would mean that 1 mL of blood was needed whereas in the DBS Within the last two years a number of articles have appeared discussing the use of dried blood in pharmacokinetic studies with several specifically focusing on children and neonates [START_REF] Spooner | Dried blood spots as a sample collection technique for the determination of pharmacokinetics in clinical studies: considerations for the validation of a quantitative bioanalytical method[END_REF][START_REF] Patel | Facilitating pharmacokinetic studies in children: a new use of dried blood spots[END_REF][START_REF] Patel | Dexamethasone quantification in dried blood spot samples using LC-MS: The potential for application to neonatal pharmacokinetic studies[END_REF][START_REF] Suyagh | Development and validation of a dried blood spot-LC-APCI-MS assay for estimation of canrenone in paediatric samples[END_REF][START_REF] Suyagh | Development and validation of a dried blood spot-HPLC assay for the determination of metronidazole in neonatal whole blood samples[END_REF]. Spooner et al [START_REF] Spooner | Dried blood spots as a sample collection technique for the determination of pharmacokinetics in clinical studies: considerations for the validation of a quantitative bioanalytical method[END_REF] have detailed the development of a DBS method for paracetamol, which they describe as being appropriate for pharmacokinetic studies, the methodology involves simple extraction followed by HPLC/MS/MS using SRM. The linear range was 25-5000 ng/mL and the technique was applied to a clinical study involving human volunteers.

Patel et al discuss the various aspects of the use of DBS sampling in order to facilitate pharmacokinetic studies in children [START_REF] Patel | Facilitating pharmacokinetic studies in children: a new use of dried blood spots[END_REF] and describe a LC-MS method for the determination of dexamethasone in DBS [START_REF] Patel | Dexamethasone quantification in dried blood spot samples using LC-MS: The potential for application to neonatal pharmacokinetic studies[END_REF], which is reported to be particularly suited to pharmacokinetic studies involving paediatric populations. Suyagh at have also described the application of DBS sampling methods for the determination of canrenone (44) levels in a pediatric population and metronidazole [START_REF] Suyagh | Development and validation of a dried blood spot-HPLC assay for the determination of metronidazole in neonatal whole blood samples[END_REF] in neonates, both methods being specifically developed for pharmacokinetic studies. The latter methodology has been utilised in what is believed to be the first published pharmacokinetic study employing DBS [START_REF] Suyagh | Metronidazole Pharmacokinetics in Preterm Neonates With Suspected Necrotizing Enterocolitis: A Population Study Using Dried Blood-Spot Sampling[END_REF].

Dried blood spot concerns

Although there have been many recent developments in DBS sampling for drug level determinations in both the adult and the paediatric/neonatal population there are a various aspects that need consideration if this technique is to become a widely applied technique in TDM and pharmacokinetic/pharmacodynamic investigations. Firstly the technique does appear to be most valuable in relation to the micro-analytical aspects, which are the focus of this review, in that many of the recent papers detail a sample volume of 30 µL or less. Despite this low volume reports indicate that the methodology is more than sensitive for the TDM/clinical investigations, LC/MS methods seem to be the norm with the added sensitivity this brings although Suyagh et al [START_REF] Suyagh | Development and validation of a dried blood spot-HPLC assay for the determination of metronidazole in neonatal whole blood samples[END_REF], along with others have effectively utilized HPLC with UV/Fluorescence detection. There is ample evidence to suggest that there has been extensive investigation of the validation of the analytical methods in terms FDA/ICH requirements [START_REF] Spooner | Dried blood spots as a sample collection technique for the determination of pharmacokinetics in clinical studies: considerations for the validation of a quantitative bioanalytical method[END_REF][START_REF] Patel | Dexamethasone quantification in dried blood spot samples using LC-MS: The potential for application to neonatal pharmacokinetic studies[END_REF][START_REF] Suyagh | Development and validation of a dried blood spot-LC-APCI-MS assay for estimation of canrenone in paediatric samples[END_REF][START_REF] Suyagh | Development and validation of a dried blood spot-HPLC assay for the determination of metronidazole in neonatal whole blood samples[END_REF], although the validations seem to have followed the general "bioanalytical" standards and not any that are specific to DBS analysis. A number of papers have detailed aspects that need to be looked into in relation to successful DBS applications in particular the sampling paper used [and whether this is pre-treated or not], the effect of different volumes of dried blood being applied to the sampling paper, the preparation of standard DBS samples in relation the patient samples, the effect of haematocrit on the sample application, the extraction of the drug from the sampling paper and the stability of samples once dried on the sample paper [START_REF] Spooner | Dried blood spots as a sample collection technique for the determination of pharmacokinetics in clinical studies: considerations for the validation of a quantitative bioanalytical method[END_REF][START_REF] Edelbroek | Dried Blood Spot Methods in Therapeutic Drug Monitoring: Methods, Assays, and Pitfalls[END_REF][START_REF] Li | Dried blood spot sampling in combination with LC-MS/MS for quantitative analysis of small molecules[END_REF][START_REF] Hoogtanders | Therapeutic drug monitoring of tacrolimus with the dried blood spot method[END_REF][START_REF] Van Der Heijden | Therapeutic drug monitoring of everolimus using the dried blood spot method in combination with liquid chromatography-mass spectrometry[END_REF]. At present it is, in the opinion of the author, impossible to specify a standard approach to the development of DBS analytical procedures for TDM and clinical studies although work to rectify is being conducted in Queens's University Belfast.

Dried blood spot use in PK studies

A second aspect of the DBS methodology is the utilisation of the data once obtained especially in the case of pharmacokinetic studies. The data generated from the DBS analysis will give whole blood concentrations whereas the majority of pharmacokinetic data will have previously been based on plasma/serum data. Various authors have developed DBS methodology and compared the results with plasma or serum values. In some instances it has been reported that the relationship between the concentrations in the two biological matrices is well correlated and of similar levels for tacrolimus and lamotrigine respectively [START_REF] Hoogtanders | Therapeutic drug monitoring of tacrolimus with the dried blood spot method[END_REF][START_REF] Soons | Lamotrigine In Dried Blood Spots By Hplc[END_REF][START_REF] Aburuz | A Simple Dried Blood Spot Assay for Therapeutic Drug Monitoring of Lamotrigine[END_REF] whereas in other instances although the results are correlated with the determined levels are somewhat different and in need of correction e.g. homocycteine [START_REF] Mccann | Measurement of total homocysteine in plasma and blood spots using liquid chromatography-tandem mass spectrometry: comparison with the plasma Abbott IMx method[END_REF], serotonin, 5hydroxy indolacetic acid and homovanillic acid [START_REF] Saracino | Chromatographic analysis of serotonin, 5-hydroxyindolacetic acid and homovanillic acid in dried blood spots and platelet poor and rich plasma samples[END_REF] and phenobarbital [START_REF] Marca | A new rapid micromethod for the assay of phenobarbital from dried blood spots by LC-tandem mass spectrometry[END_REF]. This variation in DBS/plasma[serum] concentrations has been suggested to be based on the blood/plasma ratio of the drugs and the hematocrit. The implementation of corrections is discussed in some detail by Li and Tse (48) who also indicate that the fraction of drug bound to blood should be taken into account although they do indicate possible problems in this area. Emmons and Rowland (57) have also produced an interesting commentary on the use of DBS data in place plasma/serum data for pharmacokinetic studies and suggest some possible approaches to overcoming these difficulties. It is also apparent that even with these approaches there are other important aspects that will also need addressing before the DBS sampling can be routinely used in clinical/pharmacokinetic in place of plasma/serum sampling.

Solid Phase Micro Extraction and Stir Bar Sorptive Extraction

Solid Phase Micro Extraction (SPME) and Stir Bar Sorptive Extraction (SBSE) are sample collection and clean up procedures that have been developed for a range of different areas but which are now finding applications in drug analysis. These techniques have been applied in many areas for studying environmental samples, volatile organic compounds, food and wine sampling etc but it is only in the relatively recent past that their application in drug analysis is being fully realised.

Solid Phase Micro Extraction

SPME was developed by Arthur and Pawliszyn [START_REF] Arthur | Solid phase microextraction with thermal desorption using fused silica optical fibers[END_REF] and is a sample preparation technique based on the adsorption of analytes directly from an aqueous sample onto a coated, fusedsilica fiber. This sampling technique is fast, easy to use and eliminates the use of organic solvents. The SPME fiber can be inserted into the liquid sample or suspended above the sample for headspace sampling. The fused silica fiber is coated with an appropriate coating for the extraction and in much of the early work polydimethylsiloxane (PDMS) was used; this was appropriate since many of the compounds under investigation were highly lipophilic.

Once the compound(s) have been extracted onto the coated fiber they can then be analysed by GC or HPLC, in the simplest examples the coated fiber in introduced into the heated injection port of a GC and the fiber is exposed and thermal desorption of the sample occurs, alternatively the fiber can be introduced into a small volume solvent chamber for desorption into solvent which can then be injected into a HPLC. There are a variety of factors that can be investigated in order to optimise the extraction e.g. temperature, time, desorption conditions etc., however, once optimised the extractions can be shown to produce excellent results.

Several excellent reports on the details of the basic theory and method optimization in SPME in relation to bioanalytical methodology have been published [START_REF] Ulrich | Solid-phase microextraction in biomedical analysis[END_REF](63)[START_REF] Kataoka | Recent Advances in Solid-Phase Microextraction and Related Techniques for Pharmaceutical and Biomedical Analysis[END_REF][START_REF] Kataoka | Recent advances in SPME techniques in biomedical analysis[END_REF][START_REF] Musteata | Bioanalytical applications of solid-phase microextraction[END_REF][START_REF] Musteata | Analytical methods used in conjunction with solid-phase microextraction: a review of recent bioanalytical applications[END_REF] Nagasawa et al [START_REF] Nagasawa | Rapid analysis of amphetamines in blood using head space-solid phase microextraction and selected ion monitoring[END_REF] reported the use of PDMS fibers for the extraction of nicotine, cotinine, amphetamine, methamphetamine in blood using head space SPME and GC/MS. The blood sample in a sealed vial was heated at 80 °C for 20 min and the SPME fiber was exposed for 5 min in the headspace of the vial. Following extraction the fiber was introduced into the injection port of the GC-MS where the analytes were firstly derivatised to form heptafluorobutyramide (HFB) derivatives, which were then determined, using SIM. As indicated initial studies in the area of SPME were based on PDMS coated fibers that are well suited to the extraction of highly lipophilic compounds, however, many drugs are polar and thus there has A range of drugs have been analysed using SPME including ibuprofen [START_REF] De Oliveira | Solid-phase microextraction and chiral HPLC analysis of ibuprofen in urine[END_REF] and ketamine [START_REF] Wang | Use of SPE and LC/TIS/MS/MS for rapid detection and quantitation of ketamine and its metabolite, norketamine, in urine[END_REF] in urine, antidepressants in plasma [START_REF] Alves | Analysis of tricyclic antidepressant drugs in plasma by means of solid-phase microextraction-liquid chromatography-mass spectrometry[END_REF][START_REF] Silva | In-tube solid-phase microextraction coupled to liquid chromatography (in-tube SPME/LC) analysis of nontricyclic antidepressants in human plasma[END_REF] [with the second example here demonstrating the use of in-tube SPME] and antibiotics in whole blood and plasma [START_REF] Szultka | Polypyrrole solid phase microextraction: A new approach to rapid sample preparation for the monitoring of antibiotic drugs[END_REF]. Interestingly there have been a number of applications where SPME has been employed to determine the levels of propofol [START_REF] Miekisch | Assessment of propofol concentrations in human breath and blood by means of HS-SPME-GC-MS[END_REF][START_REF] Gong | Investigation of propofol concentrations in human breath by solid-phase microextraction gas chromatography-mass spectrometry[END_REF] and fentanyl [START_REF] Wang | Intravenous fentanyl is exhaled and the concentration fluctuates with time[END_REF] in the exhaled breath of anaesthetised patients. In the above examples there has been an indication of the use of small volume sampling in SPME but not such low volumes as have been applied in the conventional and DBS sampling discussed above, additionally there are few if any specific applications in the pediatric area.

The application of this methodology to the determination of drugs in biological matrices is continuing and it is likely that it will find application in the pediatric and neonatal in the near future. One further area under study at the present is the development of SPME fibers, along with accompanying protocols, for the determination of drugs in vivo. There are several excellent papers [START_REF] Musteata | Blood sampling without blood draws for in vivo pharmacokinetic studies in rats[END_REF][START_REF] Musteata | In vivo sampling with solid phase microextraction[END_REF][START_REF] Yeung | Comparison and validation of calibration methods for in vivo SPME determinations using an artificial vein system[END_REF] in this area, which discuss the problems associated with the development of appropriate biocompatible fibers and the procedures for the validation of the analysis and the application in for example pharmacokinetic studies. To the authors best knowledge these methodologies have been applied in animal studies but as yet no human applications have been reported. and urine, plasma and brain tissues [START_REF] Unceta | Development of a stir bar sorptive extraction based HPLC-FLD method for the quantification of serotonin reuptake inhibitors in plasma, urine and brain tissue samples[END_REF]. Chaves et al [START_REF] Chaves | Stir bar sorptive extraction[END_REF] have described the determination of a range of antidepressants in plasma and hve detailed a set of procedures for the optimisation of the analysis, again researchers wishing to look at method development in this area could usefully check on this reference. Again in these examples there are little or no paediatric examples and once again the samples volumes utilised in the analyses are relatively large, however, once again it may well be possible to utilise smaller sample volumes as the methodology improves and indeed most recently a method has been developed for the determination of diclofenac in paediatric urine samples using this SBSE approach (89).

Stir Bar Sorptive Extraction

Conclusions

Thus it is clear that micro-analytical methods have become very important for paediatric/neonatal clinical/pharmacokinetic/toxicology studies. From the evidence presented above it is clear that conventional studies involving plasma/serum sampling have benefited from some of the developments in both sample preparation (SPE etc) and instrumental (MS and MS/MS) detection systems such that lower sample volumes (50-100 µL) are becoming common. The advent of DBS sampling has meant that even lower sample volumes (15-20 µL) are useable. There is also evidence that novel and automated methods are being developed for this work [START_REF] Déglon | On-line desorption of dried blood spot: A novel approach for the direct LC/MS analysis of micro-whole blood samples[END_REF][START_REF] Abu-Rabie | Direct quantitative bioanalysis of drugs in dried blood spot samples using a thin-layer chromatography mass spectrometer interface[END_REF][START_REF] Wang | Paper Spray for Direct Analysis of Complex Mixtures Using Mass Spectrometry[END_REF]. These advances in the application of DBS sampling will see the utilisation of this methodology become more common in the future although the interpretation of DBS concentrations versus plasma data will need substantial studies to investigate the factors raised by Li and Tse [START_REF] Li | Dried blood spot sampling in combination with LC-MS/MS for quantitative analysis of small molecules[END_REF] and Emmons and Rowlands [START_REF] Emmons | Pharmacokinetic considerations as to when to use dried blood spot sampling[END_REF]. SPME and SBSE have been shown to be useful in drug analysis and it is likely that these extraction methodologies will become widespread in paediatric analysis in the near future. The development of in vivo sampling techniques utilising SPME will most likely be a very important tool especially in pharmacokinetic analysis, however, the protocols will again need to developed so that comparison with presently available plasma/serum data can thoroughly

  Determination of drugs in in plasma/serum Chromatographic based micro-analytical methods are widely used for the determination of drug level concentrations in plasma and serum. In such determinations nowadays Gas Chromatography (with conventional detectors [e.g. Flame Ionisation Detector]), GC/MS or GC/MS/MS, HPLC with UV/Fluorescence detection, LC/MS and LC/MS/MS as well as Ultra Performance Liquid Chromatography (UPLC) are used. Sample preparation methods have also developed over the years and although liquid-liquid extractions (LLE) are still employed other more efficient methods such as Solid Phase Extraction (SPE), Protein Precipitation (PP)and "direct" methods have come to the fore.

  treated with 9-fluorenylmethyl chloroformate in the presence of borate buffer. The reverse phase HPLC methodology employed spectroflurometric detection and the method was linear over the range 0.1-15 mg/L. The details presented above have shown the development of micro-analytical methods utilising small volumes of biological matrices and innovative extraction methods. Despite these developments paediatric researchers have looked to develop newer methods where even lower sample volumes are employed and novel sample methods from other analytical areas have been investigated.

method only 40

 40 µL was employed and in fact only a portion [probably 10-12 µL based on an approximate calculation] of this was used for the assay. Despite the much smaller sample volume used in the DBS methodology the linear range is virtually the same.Lindkvist et al (38) have recently reported the utilisation of DBS sampling for the determination of sulfadoxine and sulfamethoxazole in children for a compliance study. The authors indicate that with malaria treatment failure may be due to drug resistance or poor compliance and thus have developed a simple DBS based sampling method to study these aspects. The DBS extraction process was a simple aqueous extraction using dilute perchloric acid, shaking for 30 min and centrifugation; this was followed by HPLC analysis incorporating UV detection. The authors indicate that the developed method was applicable for compliance studies with these two drugs.There are numerous reports of the use of DBS sampling in HIV studies in children both for the diagnosis of HIV infection and also for TDM of antiretroviral drugs. One of the tests used for testing babies born to HIV infected mothers involves a Polymerase Chain Reaction method which tests for HIV DNA this can now be accomplished using DBS (39). Amongst the plethora of papers detailing the determination of antiretroviral drugs is the recent paper by Meesters et al[START_REF] Meesters | Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots[END_REF], this describes a matrix-assisted laser desorption/ionization-triple spectrometry (MALDI-QqQ-MS/MS) for the determination of lopinavir and ritonavir concentrations in DBS samples. These authors indicate that the time for a single determination (once extracted) is only 15 seconds; which means that a whole 96 well sample tray can be analysed in approximately 17 minutes.

  been a need to develop alternative coating materials for extraction. Unceta et al[START_REF] Unceta | Simultaneous determination of citalopram, fluoxetine and their main metabolites in human urine samples by solid-phase microextraction coupled with highperformance liquid chromatography[END_REF] have reported the simultaneous determination of citalopram, fluoxetine and metabolites in human urine samples by solid-phase microextraction using HPLC. In this paper (typical of many such papers) the authors investigated the optimisation of the conditions of extraction, the factors of importance that they considered were type of fiber, speed of stirring (fixed but unstated), temperature (extraction was carried out at 21 o C), time of extraction, ionic strength and pH, organic modifier (and percentage) and desorption process. The first factor investigated was the type of fiber to be used and three were studied namely Polyacrylate, PDMS/DVB and Carbowax/Templated Resin and over the whole range of compounds the Carbowax/Templated Resin was the most appropriate. The paper describes other optimisation factors and is well worth study for an insight into this aspect for prospective users of this methodology. The analytical method developed proved most satisfactory for the determination of the drugs in urine samples.

  Stir-bar sorptive extraction is a solvent-less sample preparation method for the extraction and enrichment of organic compounds from aqueous matrices. The method is based on the same principles as solid-phase SPME and was developed by Sandra and colleagues in 1999 (81).Compared with SPME, a relatively large amount of extracting phase (PDMS) is coated on a stir bar. Solutes are extracted into the coating, based upon their [octanol/water] partitioning coefficient and upon the sample extraction medium phase ratio. The technique has been applied successfully to trace analysis in environmental, biomedical and food applications and extremely low detection limits have been reported. Most recently, SBSE has been used for extraction of drugs from various biological matrices such as urine[START_REF] Stopforth | Quantification of testosterone and epitestosterone in human urine samples by stir bar sorptive extraction--thermal desorption--gas chromatography/mass spectrometry: application to HIV-positive urine samples[END_REF][START_REF] Kawaguchi | Measurement of benzophenones in human urine samples by stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry[END_REF], plasma[START_REF] Fernandes | Determination of fluoxetine in plasma by gas chromatography-mass spectrometry using stir bar sorptive extraction[END_REF][START_REF] Queiroz | Quantification of carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbital in plasma samples by stir bar-sorptive extraction and liquid chromatography[END_REF][START_REF] Balbão | Rifampicin determination in plasma by stir bar-sorptive extraction and liquid chromatography[END_REF]

  P, Taylor PJ, Rooney F.A high-performance liquid chromatography-mass spectrometry method using a novel atmospheric pressure chemical ionization approach for the rapid simultaneous measurement of tacrolimus and cyclosporin in whole blood. V, Rowell FJ, Baker A, Laurie D, Sidki AM. A specific ELISA method for determining chloroquine in urine or dried blood spots. Bull World Health Organ. 1988; CW, Rosing H, Schellens JH, Beijnen JH. Application of dried blood spots combined with high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry for simultaneous quantification of and actinomycin-D. Anal Bioanal Chem. 2009; 394: 1171-1182. 38. Lindkvist J, Malm M, Bergqvist Y.Straightforward and rapid determination of sulfadoxine and sulfamethoxazole in capillary blood on sampling paper with liquid chromatography and UV detection. Trans R Soc Trop Med Hyg. 2009; 103: 371-376.

  E, Sandra P, David F, Cramers C. Stir bar sorptive extraction (SBSE), a novel extraction technique for aqueous samples: Theory and principles. J Microcolumn Separation. 1999; 11: 737-747. with UV detection for determination of antidepressants in plasma samples. J Chromatogr B Analyt Technol Biomed Life Sci. 2007; 850: 295-302. 89. Kole PL, Millership J, McElnay JC. Solventless extraction of diclofenac from paediatric urine samples: a stir bar sorptive extraction approach. Submitted for publication.

Figure 1 .Figure 2 .

 12 Figure 1. Structures of Topiramate[START_REF] Turner | Unlicensed and off label drug use in paediatric wards: prospective study[END_REF] and 9-fluorenylmethyl chloroformate[START_REF] Conroy | Unlicensed and off label analgesic use in paediatric pain management[END_REF]

Table 1 .

 1 List of abbreviations

	Terminology	Abbreviation
	High Performance Liquid Chromatography	HPLC
	Ultra Performance Liquid Chromatography	UPLC
	Liquid Chromatography/Mass Spectrometry/	LC/MS/MS
	Mass Spectrometry
	Dried Blood Spot	DBS
	Gas Chromatography/Mass Spectrometry/ Mass	GC/MS/MS
	Spectrometry
	Liquid Liquid Extraction	LLE
	Protein Precipitation	PP
	Solid Phase Extraction	SPE
	Limit of Quantification	LOQ
	Multiple Reaction Monitoring	MRM
	Internal Standard	IS
	Atmospheric Pressure Chemical Ionisation	APCI
	Gas Chromatography	GC
	Enzyme-Multiplied Immunoassay Technique	EMIT
	Selected Reaction Monitoring	SRM
	Solid Phase Microextraction	SPME
	Stir Bar Sorptive Extraction	SBSE

Regulation (EC) No 1901/2006 of the European Parliament and of the Council of 12 December 2006 on medicinal products for paediatric use and amending Regulation (EEC)

Pediatric Anesthesia