Clinical Biochemistry 44 (2011) 675–680

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Clinical Biochemistry

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Correlation of haloperidol levels between saliva and plasma of acutely illschizophrenic patients

Tarun Jain a,⁎, Anil Bhandari a, Veerma Ram a, Sanjay Sharma a, Manish Parakh b, Mahaveer Chand Parakh c

a Faculty of Pharmaceutical Sciences, Jodhpur National University, Jodhpur-342001 (Raj.), Indiab Department of Pediatrics, S. N. Medical College, Jodhpur (Raj.), Indiac Department of Psychiatry, S. N. Medical College, Jodhpur (Raj.), India

Abbreviations: UV-PDA, Ultra Voilet Photo-DiodeArrayDeviation; TDM, Therapeutic Drug Monitoring; HPL, HaloSaliva:Brain; S:P, Saliva:Plasma;HPLC,HighPerformanceLiquLimit of Quantification; DSM-IV-TR, Diagnostic and StatistiRevision; EDTA, Ethylene-Diamine Tetraacetic Acid; g, GraviPerformance Liquid Chromatography.⁎ Corresponding author at: Faculty of Pharmaceutic

University, Jhawar Road, Boronada, Jodhpur-342001 (Ra281416.

E-mail address: jain0291@gmail.com (T. Jain).

0009-9120/$ – see front matter © 2011 The Canadian Sdoi:10.1016/j.clinbiochem.2011.03.135

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 24 October 2010Received in revised form 1 March 2011Accepted 19 March 2011Available online 31 March 2011

Keywords:Haloperidol saliva: plasma correlationSalivary haloperidol levelsClinical monitoringNon-invasive monitoring

Objective: Clinical usefulness of monitoring haloperidol in salivary samples based on plasma:salivacorrelation.

Design and Methods: Plasma and saliva samples of schizophrenic patients [N=105] were analyzed byhighly sensitive reverse phase liquid chromatographic method to measure haloperidol at 240 nm using UV-PDA detector. Mobile phase consist of acetonitrile and water [50:50], pH 2.5 (0.1% acetic acid and 0.05 MKHPO4) at flow rate 1.4 mL/min. Method was linear over 3–200 ng/mL.

Results: Observed therapeutic range was 5–19 ng/mL [11.66±3.97] and 17–54 ng/mL [27.52±11.51] forplasma and saliva respectively. Mean S:P was found to be 2.36.

Conclusion: Current study showed significantly high correlation [r=0.93, pb0.0001] betweenhaloperidol levels in saliva and plasma with linear relationship. It is therefore concluded that monitoring

of salivary concentration can be a clinically beneficial substitute. Patients showing clinical improvement[N=90] were within salivary concentration range of 17–54 ng/mL, which can be an appropriate steady statemonitoring range for haloperidol in saliva.

© 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction

Therapeutic drug monitoring (TDM) is a multidisciplinary areathat allows clinicians, clinical pharmacologists and laboratory spe-cialists to collaboratively optimize patient drug therapy. Althoughdrug measurement of blood or plasma is essential for TDM, the factthat some drugs secrete in saliva has led to the use of saliva for TDM[1–4].

In clinical conditions where protein binding varies, salivary levelmay be more closely related to therapeutically active drug levels (freeform) than plasma level as salivary level may accurately reflect thefree drug level [1–4]. Drugs which are not ionizable or remain un-ionized at salivary pH like phenytoin, carbamazepine, theophylline,lamotrigine and haloperidol (HPL) are candidates for salivarymonitoring [5,6].

Detector; RSD, Relative Standardperidol; P:S, Plasma:Saliva; S:B,idChromatography;LLQ, Lowercal Manual, Fourth Edition, Textty; RP-HPLC, Reverse Phase-High

al Sciences Jodhpur Nationaljasthan), India. Fax: +91 2931

ociety of Clinical Chemists. Publish

The use of saliva to determine drug concentrations has severaladvantages over blood as it involves non-invasive procedure; easysampling and frequent monitoring thus may be more acceptable tothe patient and convenient for the physician. Salivary monitoring hassome disadvantages like oral contamination, can only be used forselective drugs in unionized form and can be influenced by pH. Incertain situations, where the concentration of protein bound drug isconsiderable—determination of salivary drug concentration can be agood measure. Significant correlations between salivary and plasmaconcentrations have been shown for chlorpromazine, HPL, diazepam,and lithium [7–11].

In general, anti-psychotics are administered orally at very lowdoses and are widely metabolized in body, therefore the plasma drugconcentration is very low [12,13]. TDM of anti-psychotic drugs hasproven valuable for determining poor compliance of patients andaddressing challenges associated with considerable genetic variabilityin their metabolism. Thus, in order to conduct the pharmacology,toxicology and clinical TDM of anti-psychotics, a highly selective andsensitive bioanalytical method is much needed [14].

HPL (CAS 52-86-8, Fig. 1) is chemically 4-[4-(p-chlorophenyl)-4-hydroxypiperidino]-4'-fluorobutyrophenone. It is light yellow crys-talline powder, have melting point 149–153 °C, pH 2.8-3.6 and pKa of8.3. It is soluble in solvents like glacial acetic acid, chloroform andalcohol but almost insoluble in water. It is used for the symptomaticmanagement of psychotic disorders. Drug therapy is integral to the

ed by Elsevier Inc. All rights reserved.

Fig. 1. Haloperidol.

676 T. Jain et al. / Clinical Biochemistry 44 (2011) 675–680

management of acute psychotic episodes and accompanying violentbehavior in patients with schizophrenia, generally required for long-term stabilization to improve symptoms between episodes andminimize the risk of recurrent acute episodes [12,15].

TDM of HPL is clinically useful as treatment requires individualizedoptimization due to narrow therapeutic window and large inter-individual variability of its pharmacokinetics [14–17]. Despite theaforementioned advantages of salivary monitoring, clinical usefulnessof TDM for HPL using salivary samples based on S:P correlation is yetto be validated [18].

Till now very few studies have been reported in humans thatcompared HPL concentration in saliva and blood. Yamazumi andMiura [8] reported that the concentrations of HPL in saliva correlatewell with plasma concentrations while Zohar et al. [11] failed tocorrelate any significant relationship between saliva and blood HPLlevels. Dysken et al. [19] observed good correlation of HPL levels inplasma and saliva but the sample size of study was very small.

In a pioneer study by Noriyasu et al., correlation of HPL levelbetween saliva:plasma [S:P] and saliva:brain [S:B] were established inrats. To stimulate secretion, the chorda (parasympathetic) andsuperior cervical ganglion (sympathetic) were electrically stimulated,and pilocarpine was administered. Findings of this study indicate thatsalivary HPL levels reflect levels in brain more precisely than bloodHPL levels [18].

Various methods currently used for TDM of HPL in plasma sampleshave someor other disadvantages like: longer run time [8,9], solid phaseextraction (time consuming and expensive) [8], narrow linearity range(eventually not suitable for toxicological studies) [8,10], high matrixvolume [11] and greater lower limit of quantification (LLQ) (unfit tocover entire therapeutic range effectively) [20–24].

Few analytical procedures have been described previously fordetermination of antipsychotics using HPLC, but these methods aremainly used for screening purposes and none of the methodapproaches toward S:P correlation [5–7].

The aim of this study was to develop and validate a selective andsensitive RP-HPLC method and to explore S:P correlation whichfacilitates future non-invasive clinical monitoring of HPL.

Materials and methods

Study population and sample size

The current study was conducted in collaboration with MathuradasMathur Hospital (Site 1), Jodhpur Hospital and Multi Specialty Centre(Site 2) and Faculty of Pharmaceutical Sciences (Site 3), Jodhpur.Approval was obtained from ethics committee of site 1 and 2; sampleanalysiswas performed at site 3. Patients [N=105]were enrolled basedon inclusion criteria like: willing to participate in study, prescribedwithoral HPL, acutely ill, having at least one follow up visit, between 18 and70 years of age, no hepatic or renal failure while patients suffering from

other chronic disease/infections, on drug regimen known to alter themetabolism of studied drugs were excluded. Written informed consentwas obtained from patient/guardian enrolled in study. On the basis ofDSM-IV TR criteria, patients were mainly diagnosed as schizophrenic[N=54], bipolar disorders [N=13], schizoaffective disorder [N=27],delusional disorder [N=9] and organic mental disorder [N=2].

Chemicals and standard solutions

HPL (purity 99.9%) was gifted by Zydus-Cadila Pharmaceutical Pvt.Ltd, Ahemdabad (Gujarat) and loratidine [IS, selected on the basis ofsimilar solubility profile and structural resemblance] from AlembicPharmaceuticals, Baddi, Himachal Pradesh. Blank human plasmasamples were made available by Paras Blood Bank while saliva wascollected from six healthy individuals. All reagents (Merck) used wereof HPLC grade except acetic acid and KHPO4 which were of analyticalgrade. Water was glass triple-distilled and further purified with a0.45 μ filtration membrane using vacuum pump.

Equipments

The HPLC system used was Cecil ADEPT CE 4700 solvent deliverysystem along with system controller (CE 4900), a UV–PDA detector(CE 4200) operated at wavelength of 240 nm, a degasser and a dataprocessor (all from Cecil, England, United Kingdom).

Chromatographic conditions

The method was developed and validated using C18-ODS column(Thermo, 250 mm×4.6 mm I.D., 5 μm particle size) protected by aguard column (1 cm×4 mm I.D., 5 μm particle size). A mixture ofacetonitrile and water containing 0.1% acetic acid (pH adjusted to 2.5using 0.05 M KHPO4) [50:50 v/v] was used as the mobile phase(isocratic separation). The detection wavelength was 240 nm. Mobilephase was filtered, degassed and pumped at flow rate of 1.4 mL/min.

Preparation of sample solution and stock solution

1.0 mg of pure HPL was weighed accurately and transferred intoround bottom flask. 100 mL of methanol was added and sonicated for10 min to dissolve the drug completely. Now, solution was filteredthrough a 0.45 μmNylon 66membrane filter using vacuum pump andfurther diluted using methanol to give stock solutions 10 μg/mL. Stocksolution of IS was prepared in similar manner. Working standards ofHPL (3–100 ng/mL) and IS (100 ng/mL) were prepared by serialdilution of the stock solution in methanol and stored at 4 °C.

Extraction of HPL from human plasma and saliva

Extraction from plasma and saliva was carried out using liquid-liquid extraction technique. 300 μL of human plasma samplewasmixedwith 50 μL of IS (100 ng/mL) and vortexed for 1 min. Then 300 μL ofisopropyl alcohol (HPLC grade, Merck) was added and centrifuged at5590g for 5 min. Clear organic layer was separated and evaporated inturbo vap LV Evaporator (Zymark, Hopkinton, MA, USA) at 50 °C understream of nitrogen. Dried residue was then reconstituted with 300 μLmobile phase and 100 μL injected for HPLC analysis.

Method validation

Current method was validated as per FDA guidelines forbioanalytical method validation [25].

Specificity and selectivitySpecificity of method was determined by analyzing six replicates of

blank human plasma/saliva obtained from six different sources. Each

Fig. 2. Spectrum of haloperidol in patient plasma.

677T. Jain et al. / Clinical Biochemistry 44 (2011) 675–680

blank sample was tested for interference and selectivity was ensured atlower limit of quantification [LLQ]. Other possible interfering substanceslike co administeredmedicines, blood components like hemoglobin etc,metabolites and excipients were investigated by direct injection ofstandards onto the HPLC column and measurement of their respectiveretention times. The results are represented in Table 1 and Fig. 2.

Linearity and range of calibration curveCalibration curves (n=5) of different concentration range were

determined in biological matrix (plasma and saliva) by spiking matrixwith known concentrations of analyte including a blank sample(matrix sample without internal standard), zero sample (matrixsample with internal standard), and eight non-zero samples coveringexpected range, including LLQ. The results of studied concentrationsfor each calibration curve studied are represented in Linearity andrange of calibration curve.

LOD and LLQ . Signals of blank plasma and saliva samples werecompared with samples of known low concentrations of analyte inrespective matrix. Next, signal to noise ratio at which analyte can bereliable quantified (3:1 for LOD and 10:1 for LLQ) was determined.Procedure for LOD and LLQ was replicated 6 times. The results arerepresented in LOD and LLQ.

Accuracy and precisionChoosing 3 concentrations from LLQ, 2–5 times LLQ, 0.5 times ULQ,

ULQ and above ULQ; each spiked sample of plasma and saliva wasanalyzed in 6 replicates for within run and between run accuracy andprecision. The results are represented in Table 2.

RecoveryRecovery experiments were performed by comparing results of

extracted samples at low, medium, and high concentration with un-extracted standards (100%) injected directly into HPLC system. Eachobservation was determined in triplicate. Recovery of I.S. wasevaluated by comparing mean peak areas of extracted samples ofplasma and saliva to mean peak areas of reference solutions (un-extracted) for same concentration. The results are represented inTable 3.

StabilityStability of HPL in plasma and saliva matrices was studied at three

different stability conditions: short term, freeze–thaw, and long term.Stability was determined by analyzing low, medium and highconcentration samples spiked in plasma and saliva matrices. Shortterm stability was carried out by keeping samples for approximately6 h at room temperature. Freeze–thaw stability was determined overthree freeze-thaw cycles, by thawing at room temperature for 2–3 hand freezing for 12 h in each cycle. Long term stability was tested afterstorage at −80 °C for 30 days. For each concentration and storage

Table 1Retention times of drugs tested for interference in the assay.

Drugs Retention time (min)

Diazepam 08:43Nitrazepam 06:56Olanzapine 07:31Haloperidol 02:35Aripiprazole 09:29Alprazolam 10:25Loratidine 03:53Clozapine 07:56Carbamazepine 19:00Sodium Valproate. 22:43

condition, six replicates were analyzed in one set of batch. The resultsare represented in Table 4.

Sample pretreatment; for evaluation of therapeutic levels

Blood and saliva samples were collected in morning between08:00 and 09:00, just before next dose to ensure the trough levels ofHPL. For facilitating the secretion of saliva, a drop of citric acid wasplaced into subject's mouth 30 s before collection. Blood sampleswerecollected in EDTA vaccutainers and further processed at 5590g for15 min to separate plasma. Salivary samples were collected in widemouth plastic containers. The collected plasma and saliva were thenstored at −80 °C till analysis. For quantitative estimation, a reliablepretreatment of biological samples (n=105) was done according toprocedure discussed in Extraction of HPL from human plasma andsaliva.

Table 2Accuracy and precision studies (n=6) of HPL in plasma and saliva.

Parameters Within run Between run

AddedConc.

NominalConc.a

[Mean±SD]

Accuracy(%)

Precision(%RSD)

NominalConc.a

[Mean±SD]

Accuracy(%)

CV%

Plasma matrix3 2.86±0.061 95.33% 2.144 2.71±0.076 90.27% 2.7910 10.61±0.49 106.11% 4.611 10.30±0.61 103.00% 5.9230 28.62±1.47 95.41% 5.1481 27.95±1.65 93.19% 5.91

Saliva matrix10 9.23±0.29 92.30% 3.11 8.89±0.47 88.90 5.225 23.23±1.67 92.92% 7.19 22.81±1.78 91.24 7.8100 88.87±7.29 88.87% 8.21 87.76±8.31 87.76 9.47

a Concentrations are expressed in ng/mL.

Table 3Recovery studies of HPL (n=3) in plasma and saliva.

Added Conc.a Nominal Conc. [Mean±SD] % Recovery CV %

HPL plasma concentrationa

3 2.85±0.08 95% 2.8810 10.68±0.60 106.80% 5.6030 28.03±1.42 93.43 5.07

HPL saliva concentrationa

10 9.08±0.31 90.8 3.4125 22.97±1.87 91.88 8.14100 92.59±5.81 92.59 6.27

Internal standard concentrationa

10 8.83±.45 88.32% 5.12

a Concentrations are expressed in ng/mL.

678 T. Jain et al. / Clinical Biochemistry 44 (2011) 675–680

Statistical analysis

Paired t-test was applied as statistical tool for evaluation ofdifferences for whole saliva versus plasma concentrations. A t-testdependent correlation was used to compare correlation coefficient.

Results

In the current study, 105 patients on HPL [86 males, 19 females]within 18–70 years of age [35.12±12.34] were studied. HPL doseranged from 20 to 30 mg/day. All patients were on one or moreconcomitant medicines like diazepam, nitrazepam, olanzapine,aripiprazole, alprazolam, clozapine, carbamazepine and sodiumvalproate.

Selectivity and specificity

HPL and IS were well separated from co-eluted components andthere was no interference from endogenous material, observed atretention time of both HPL and IS (Table 1 and Fig. 2). The peaks wereof good shape, completely resolved from plasma components. It

Table 4Stability studies of HPL (n=6) in human plasma and saliva.

Sample Conc.a Plasma Conc.a % Accuracy CV %

Short term stability (6 h)3 3.21±0.11 107 3.4410 10.13±0.78 101.30 7.7230 26.87±1.23 89.56 4.55

Long term stability (−80 °C, 1 month)3 2.67±0.21 89 7.9510 9.13±0.91 91.30 9.9930 26.53±1.41 88.40 5.32

Freeze and thaw cycles3 2.91±0.132 97 4.5310 9.77±0.67 97.70 6.8730 27.90±1.44 93 5.16

Sample Conc.a Saliva Conc.a % Accuracy CV %

Short-term stability (6 h)10 8.89±0.47 88.90 5.2925 24.03±.88 96.12 3.66100 96.85±3.22 96.85 3.32

Long term stability (−80 °C, 1 month)10 8.52±0.821 85.2 9.6425 23.17±2.34 92.68 10.10100 89.98±5.97 89.98 6.63

Freeze and thaw cycles10 9.11±0.49 91.10 5.3825 23.81±0.82 95.24 3.46100 96.06±2.19 96.06 2.28

a Concentrations are expressed in ng/mL.

indicates that the developed method was highly selective for thematrices like plasma and saliva. The developed method was veryspecific as inferred from the retention time data of co-administereddrugs like diazepam, nitrazepam, olanzapine, aripiprazole, alprazolam,clozapine, carbamazepine and sodium valproate.

Linearity and range of calibration curve

For both plasma and saliva matrix, five calibration curves werestudied at different concentrations of calibrators in each set: 1, 5, 10, 25,50, 100, 150 and 200 ng/mL; 3, 5, 10, 25, 50, 100, 150, 200 ng/mL; 5, 10,30, 50, 75, 100, 150, and 200 ng/mL; 10, 20, 30, 50, 75, 100, 150, and200 ng/mL and 10, 30, 50, 75, 100, 125, 150, and 200 ng/mL. Thedeveloped and validatedmethodwas linear over range of 3–200 ng/mLwith coefficient greater than 0.998.

LOD and LLQSignal of blank samples was compared with samples containing

known low concentrations of the analyte. The LOD and LLQ in plasmaand saliva were found to be 1.1±0.12 ng/mL; 3.0±0.12 ng/mL and1.0±0.06 ng/mL; 3.1±0.14 ng/mL respectively.

Accuracy and precision

Accuracy and precision values for within and between run studiesat low, medium and high quality control concentrations of HPL inplasma and saliva were within acceptable limits (Table 2). The resultalso indicated that assay method was reproducible, accurate andprecise for analysis of HPL.

Extraction recovery

Over the concentrations studied, minimum extraction recovery ofHPL and IS in plasma and saliva was greater than 87%. The details ofrecovery study are represented in Table 3.

Stability

Stability of drug spiked at three QC levels evaluated for: short term(6 h), freeze–thaw (3 cycles), and long term (30 days). The results(Table 4) confirmed that HPL was quite stable in human plasma andsaliva for about one month when stored in frozen state (−80 °C).

Clinical application of developed and validated method

This validated high-performance liquid chromatographic methodusing a simple mobile phase was successfully applied for clinicalmonitoring of HPL in psychiatric patients. Typical saliva and plasmalevel chromatograms of patient taking HPL are given in Fig. 3 [A] andFig. 3 [B] respectively. Negligible effect on drug concentration (b1%)was observed in stimulated and non-stimulated salivary sampleswhen observed in same subjects (n=6).

In 3 cases, data points were eliminated from the study as observedfrom a well-established fact that carbamazepine can alter assay resultof HPL by reducing plasma concentration. 12 patients showedabnormal levels of saliva or plasma concentrations which were eithersub-therapeutic or toxic; therefore final correlation was establishedby analyzing data from 90 patients.

The therapeutic range of HPL in saliva and plasma [N=90] wasfound to be 17–54 ng/mL [27.52±11.51] and 5–19 ng/mL [11.66±3.97] respectively. Theminimum,mean, andmaximumS:Pwas found tobe 1.54, 2.36 and 2.79 respectively. Median saliva and plasmaconcentration was 36.70 and 13.68 ng/mL respectively. Fig. 4 illustratescorrelation between saliva and plasma, found to be 0.93 [at 95% CI,pb0.0001].

79

47

14

-18

-50

00:00 06:0000:00 06:00

54

19

-16

-50

Abs

orba

nce

[mA

]

Abs

orba

nce

[mA

]Hal

oper

idol

Lora

tidin

e

Hal

oper

idol

Lora

tidin

e

A B

Fig. 3. [A] Chromatogram of plasma sample from patient on Haloperidol (11.38 ng/ml) spiked with IS Loratidine. [B] Chromatogram of saliva sample from patient on Haloperidol(32.82 ng/ml) spiked with IS Loratidine.

679T. Jain et al. / Clinical Biochemistry 44 (2011) 675–680

Discussion

In the current study, a significant correlation (r=0.93) wasobserved between salivary and plasma levels. The pKa of haloperidol(8.3) keeps it unionized at salivary pH and its lipophilic naturefacilitates excretion in saliva through passive diffusion. Variableswhich influence this type of transport are pH, pKa, lipid solubility andmolecular weight of drug. The aforementioned statement also holdstrue for certain other drugs like phenytoin, carbamazepine, lamo-trigine and theophylline.

Previous studies showed similar correlation but sample size wastoo small to be applicable on large group of population [19]. Study byYamazumi [8] showed a less significant correlation while Zohar andassociates failed demonstrate any correlation between serum andsalivary concentration of HPL.

Animal study by Noriyasu et al. [18] proved that correlationbetween saliva and brain was higher than that between plasma and

Saliva-Plasma Correlation of Haloperidoly = 2.6939x - 0.2426

R = 0.863

0.00

10.00

20.00

30.00

40.00

50.00

60.00

0.00 5.00 10.00 15.00 20.00 25.00

Plasma Concentration (ng/ml)

Sal

ivar

y C

on

cen

trat

ion

(n

g/m

l)

2

Fig. 4. Plasma and saliva concentration correlation (r=0.93) of HPL (n=90).

saliva; results suggesting that HPL was specifically secreted from thestriated duct system, and salivary concentrations reflect levels in brainmore precisely than plasma. Further, high correlation of HPL levelsbetween saliva and brain [18] suggests salivary HPL levels couldindicate proportional levels in brain. An interesting study by JohannesKornhuber et al. [26] showed that HPL concentration in human braintissue was 10–30 times higher than optimal serum concentrations inschizophrenic patients.

Current study with comparative large sample size (N=90)showed a significant correlation (r=0.93) between saliva and plasmaconcentration which appears that monitoring of salivary concentra-tion can be suitable and clinically beneficial substitute of plasmamonitoring for patients on HPL.

Studies by Noriyasu et al. and Kornhuber et al. [18,26] confirmedhigh brain HPL concentration and its correlation with both salivaryand plasma concentration. In the present study, mean saliva:plasmaratio was found to be 2.3 folds higher than plasmawhich will improvethe diagnostic importance of salivary monitoring in psychiatricpatients. Patients who showed clinical improvement [N=90] hadsalivary concentration in the range of 17–54 ng/mL which can be anew steady state range for monitoring clinical improvement inschizophrenic patients receiving HPL in spite of routine plasma steadystate range between 5 and 19 ng/mL.

This study involved development, validation and clinical applica-tion of analytical method for routine estimation of HPL. Although HPLis not used in chronic conditions it is a drug of choice in acute casesbecause of its low cost and traditional prescription practice. Thecontributory findings of current study can be beneficial for patients onHPL. Current study developed and validated a time sparing andeconomic method using simple lab instruments like RP-HPLC. Clinicalapplications of this method will be of definite use in TDM andtoxicological management in patients on HPL.

Acknowledgments

The authors would like to thank Zydus-Cadilla and AlembicPharmaceuticals for providing pure haloperidol and loratidine usedfor method development and validation. The authors also thank ParasBlood Bank, Jodhpur for facilitating the sample storage during entirestudy duration.

680 T. Jain et al. / Clinical Biochemistry 44 (2011) 675–680

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