Simple and validated HPLC-UV analysis of levetiracetam in deproteinized plasma of patients with epilepsy

We present a simple, fast and validated method for the determination of the new generation antiepileptic drug (AED) levetiracetam (LEV) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma sample (500 microL) pretreatment was based on simple deproteinization by methanol spiked with the internal standard (I.S.). HPLC analysis was carried out on a Synergi 4-microm Hydro-RP, 150 mm x 4 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5) and acetonitrile (94:6, v/v) at an isocratic flow rate of 1.5 mL/min. The UV detector was set at 205 nm. Calibration curves were linear (mean correlation coefficient=0.999) over a range of 4-80 microg/mL. The quantitation limit was 2 microg/mL and the absolute recovery was >90% for LEV and the I.S. Both intra and interassay precision and accuracy were lower than 7.5%. The chromatographic run lasted 13 min. The present procedure omitting expensive solid phase or time-consuming liquid-liquid extraction and drying steps is cheaper, faster and simpler than mostly published analytical methods for levetiracetam. Applied to a large population of patients with epilepsy this assay proved very practical in our therapeutic drug monitoring setting (TDM).     

Relevance of a combined UV and single mass spectrometry detection for the determination of tenofovir in human plasma by HPLC in therapeutic drug monitoring

A sensitive high-performance liquid chromatography method coupled to UV and single mass spectrometry (MS) detection was developed for the determination of tenofovir in human plasma. A solid phase extraction procedure (Bond-Elut C18 Varian cartridges) provided high extraction efficiency (91% for tenofovir and 68.8% for the internal standard, 3-methylcytidine). An atlantis-dC-18 analytical column is used with an isocratic mode elution of a mixture (pH 2.5) of ammonium acetate/methanol (98.5:1.5, v/v). Detection was performed at 260 nm and by using the ion at m/z 288. The signals from both detectors were validated over the range of 10-1000 ng mL(-1) and were found to be linear, accurate and precise. At the lowest limit of quantification, 10 ng mL(-1) for UV and 5 ng mL(-1) for MS, the average coefficient of variation was 6.9 and 3.9%, respectively. To investigate the potential of the validated method for clinical studies, more than 170 samples from HIV-infected adult patients were then analyzed with this assay. A good correlation was observed between the results obtained with both detectors. However, in several cases discordant results were observed between UV and MS detections. Therefore, tenofovir can sometimes suffer from interferences using either UV or single MS detection. We concluded that the double detection allows to obtain a more specific quantification of tenofovir. The present assay is sound and can be used for therapeutic drug monitoring allowing a higher reliability of the results which are transmitted to the medical team.     

Determination of helicidum and its metabolites in dog plasma by LC/UV/MS/MS and its application to pharmacokinetic studies

A simple, rapid and reliable method was developed for the identification and quantification of helicidum and its metabolites in beagle dog plasma by liquid chromatography/ultra-violet/electrospray ionization-ion trap mass spectrometry (LC/UV/ESI-ITMS). Two metabolites were identified by MS: formylphenyl-O-beta-d-pyranosyl alloside (I) and hydroxylmethylphenyl-O-beta-d-pyranosyl alloside (II). UV was used for concentration determination with the wavelength of 270 nm. Liquid-liquid extraction was used and the extraction recovery exceeded 90%. Kromacil C(18) column (5 microm, 4.6mm i.d. x 250 mm) was used as the analytical column. Linear detection responses were obtained for helicidum concentration ranging from 1.76 x 10(-4) to 70.4 x 10(-4) micromol/mL (0.050-2.00 microg/mL). The precision and accuracy data, based on intra- and inter-day variations over 3 days, were less than 5%. The limit of determination and quantitation (LOD, LOQ) for helicidum was 0.010 and 0.030 microg/mL, respectively. Pharmacokinetic data of helicidum and the two metabolites were obtained with this method after administration of intravenous injection and a single oral dose of tablets to six beagle dogs, respectively.     

Isocratic solid phase extraction-liquid chromatography (SPE-LC) interfaced to high-performance tandem mass spectrometry for rapid protein identification

Reversed-phase liquid chromatography interfaced to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) allows analysis of very complex peptide mixtures at great sensitivity, but it can be very time-consuming, typically using 60 min, or more, per sample analysis. We recently introduced the isocratic solid phase extraction-liquid chromatography (SPE-LC) technology for rapid separation (~8 min) of simple peptide samples. We now extend these studies to demonstrate the potential of SPE-LC separation in combination with a hybrid linear ion trap-Orbitrap tandem mass spectrometer for efficient analysis of peptide samples in proteomics research. The system performance of SPE-LC-MS/MS was evaluated in terms of sensitivity and efficiency for the analysis of tryptic peptide digests obtained from samples consisting of up to 12 standard proteins. The practical utility of the analytical setup was demonstrated by the analysis of <15 microg depleted human serum proteome by a combination of SDS-PAGE and SPE-LC-MS/MS. A total of 88 unique gene products spanning 3 orders of magnitude in serum protein concentration were identified using stringent database search criteria.     

Generic solid phase extraction-liquid chromatography-tandem mass spectrometry method for fast determination of drugs in biological fluids

A generic method was developed for the fast determination of a wide range of drugs in serum or plasma. The methodology comprises generic solid-phase extraction, on-line coupled to gradient HPLC with tandem mass spectrometric detection (SPE-LC-MS/MS). The individual components of the SPE-LC-MS/MS system were optimized in an integrated approach to maximize the application range and minimize the method development time. The optimized generic SPE-LC-MS/MS protocol was evaluated for 11 drugs with different physicochemical properties. Good quantification for 10 out of 11 of the pharmaceuticals in serum or plasma could be readily achieved. The quantitative assays gave recoveries better than 95%, lower quantification limits of 0.2-2.0 ng/ml, acceptable precision and accuracy and good linearity over 2-4 orders of magnitude. Carry-over was determined to be in the range of 0.02-0.10%, without optimization.     

Study of the determination and pharmacokinetics of bufadienolides in dog's plasma after administration of Liu-Shen-Wan by high performance liquid chromatography time-of-flight mass spectrometry

A sensitive and reliable high performance liquid chromatography-tandem time-of-flight mass spectrometry method (HPLC/TOF MS) has been developed to determine three active bufadienolides from Liu-Shen-Wan (LSW) in dog's plasma. Enhanced selectivity and sensitivity in comparison with traditional HPLC/DAD method could be obtained through this method. Bufodienolides could be well separated and distinguished from its nominally isobaric endogenous components by HPLC/TOF MS, with the linear calibration range covering from 0.5 ng/mL to 100 ng/mL and Limit of Detection (LOD) being about 0.15 ng/mL. This method was also proved to be quite stable, with the intra-day precision and the inter-day precision results being lower than 6.39% and 7.44%, respectively. Meanwhile HPLC/TOF MS was successfully used in the pharmacokinetic study of LSW. For resibufogenin, the major pharmacokinetic parameters AUC0-t, Cmax and t1/2alpha were 160.72+/-21.97 ng/mL min, 2.35+/-0.71 ng/mL and 20.74+/-5.89 min, respectively, and for bufalin the corresponding parameters were 55.55+/-7.55 ng/mL min, 0.91+/-0.15 ng/mL and 25.45+/-13.28 min, respectively.     

A sensitive multidimensional method for the detection,characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection

Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window. The reduction of sample preparation steps through the incorporation of multidimensional techniques has afforded analysts more efficient methods to assess trace drug species. Multidimensional methods coupling size-exclusion and reversed phase liquid chromatography with ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity and can limit method optimization. The current study addresses these challenges with a multidimensional method that is specific, sensitive, and enables method control in both dimensions via coupling of an on-line solid phase extraction column to RPLC with mass spectral detection (SPE-RPLC/MS). The proposed method was evaluated using an antibody-fluorophore conjugate (AFC) as an ADC surrogate to brentuximab vedotin and its associated parent maleimide-val-cit-DSEA payload and the derived N-acetylcysteine adduct formed during the conjugation process. Assay sensitivity was found to be 2 orders more sensitive using MS detection in comparison to UV-based detection with a nominal limit of quantitation of 0.30 ng/mL (1.5 pg on-column). Free-drug species were present in an unadulterated ADC surrogate sample at concentrations below 7 ng/mL, levels not detectable by UV alone. The proposed SPE-RPLC/MS method provides a high degree of specificity and sensitivity in the assessment of trace free drug species and offers improved control over each dimension, enabling straightforward integration into existing or novel workflows.     

Application of a sensitive liquid chromatographic/tandem mass spectrometric method to pharmacokinetic study of nalmefene in humans

A sensitive, specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed and validated for quantification of nalmefene in human plasma. An aliquot of 200 microL plasma sample was simply precipitated by 400 microL methanol. Separation of nalmefene and the internal standard hydromorphone from the interferences was achieved on a C(18) column followed by MS/MS detection. The analytes were monitored in the positive ionization mode with a TurboIonspray source. The method had a total chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 10-5000 pg/mL. The lower limit of quantification (LLOQ) was 10 pg/mL. The intra- and inter-day precision was less than 10.1% determined from QC samples at concentrations of 30, 300 and 4500 pg/mL, and the accuracy was within +/-3.4%. As the method was more sensitive (10 times higher) than those reported previously, we investigated the pharmacokinetics of nalmefene in healthy volunteers after a single intravenous injection of low dose (30 microg) of nalmefene hydrochloride for the first time.     

Analysis of antibiotics in urine and wipe samples from environmental and biological monitoring--comparison of HPLC with UV-, single MS- and tandem MS-detection

Results of the simultaneous determination of the structurally different antibiotics cefazoline, cefotiame, cefuroxime, chloramphenicol, ciprofloxacin, ofloxacin, sulfamethoxazole and trimethoprim from environmental and biological monitoring using high-performance liquid chromatography with UV, single mass and tandem mass spectrometry were compared. For sample enrichment and clean-up a SPE method using bakerbond C18 cartridges was developed. Mean recovery rates were above 70%. Because of the complex urine matrix, only the wipe samples could be analyzed by UV-detection. However, UV-detection and single MS-detection are useful for control measurements after spillage, e.g. (LOD=1-2 ng/cm(2)). Samples from biological monitoring of occupational uptake should be analyzed by LC-MS/MS. The limits of detection (LOD) in urine ranged from 0.4 to 70 microg/L for LC-MS and 0.01 to 0.9 microg/L for LC-MS/MS detection. The limits of detection in wipe samples ranged from 0.003 to 0.13 ng/cm(2).     

Separation and determination of dexamethasone sodium phosphate in cochlear perilymph fluid by liquid chromatography with ultraviolet monitoring and electrospray ionization mass spectrometry characterization

The method for separation and determination of dexamethasone sodium phosphate (DexP) in cochlear perilymph fluid (CPF) of cavy was developed using HPLC with ultraviolet (UV) monitoring and electrospray ionization/mass spectrometry (ESI/MS) identification. The quantitative determination of DexP in CPF was achieved by HPLC with UV detection at 245 nm. The separation was carried out on a Phenomenex ODS(3) column ( 250 mm x 4.6 mm i.d., 5 microm) with the mobile phase of acetonitrile-5mmol/l ammonium acetate (23:77 (v/v)) at a flow rate of 1.0 ml/min. DexP was baseline separated from the matrices of CPF blanks within 15 min. The linearity ranged from 0.5 to 50 microg/ml. The limit of detection was 0.10 microg/ml. The recovery ranged from 98.5 to 100.8%. The relative standard deviations (R.S.D.s) of intra- and inter-day peak area were between 0.7-1.3 and 1.2-3.5%, respectively. Both full scan MS and MS2 of DexP with positive and negative polarity were obtained and elucidated. The specific ions were chosen to characterize DexP in the CPF sample. Using the proposed HPLC-UV-ESI/MS method, the concentration of DexP in CPF samples after both vein and middle ear injections were determined, and the relationships between concentration and time were obtained. This method offered reference data for clinical investigation of DexP to cure ear diseases.     

Determination of inositol hexanicotinate in rat plasma by high performance liquid chromatography with UV detection

A HPLC method with UV detection at 262nm was developed to analyze inositol hexanicotinate in rat plasma. Plasma samples were extracted with an equal volume of acetonitrile, followed by dilution with mobile phase buffer (5mM phosphate buffer, pH 6.0) to eliminate any solvent effects. Inositol hexanicotinate and the internal standard (mebendazole) were separated isocratically using a mobile phase of acetonitrile/phosphate buffer (35:65, v/v, pH 6.0) at a flow rate of 1.0mL/min and a reverse-phase XTerra MS C(18) column (4.6mmx150mm, 3.5microm). The standard curve was linear over a concentration range of 1.5-100.0microg/mL of inositol hexanicotinate in rat plasma. The HPLC method was validated with intra- and inter-day precisions of 1.55-4.30% and 2.69-21.5%, respectively. The intra- and inter-day biases were -0.75 to 19.8% and 2.58-22.0%, respectively. At plasma concentrations of 1.5-100microg/mL, the mean recovery of inositol hexanicotinate was 99.6%. The results of a stability study indicated that inositol hexanicotinate was unstable in rat plasma samples, but was stable in acetonitrile extracts of rat plasma for up to 24h at 4 degrees C. The assay is simple, rapid, specific, sensitive, and reproducible and has been used successfully to analyze inositol hexanicotinate plasma concentrations in a pharmacokinetic study using the rat as an animal model.     

A sensitive assay for simultaneous determination of plasma concentrations of valganciclovir and its active metabolite ganciclovir by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of valganciclovir and its active metabolite ganciclovir in human plasma. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on an Aquasil C18 column (50 mm x 2.1mm, 5 microm). A linear gradient mobile phase between 0.02% formic acid and methanol was used. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 4 to 512 ng/mL for valganciclovir and from 0.1 to 12.8 microg/mL for ganciclovir, were fitted to a 1/x weighted quadratic regression model. The method was proved to be accurate, specific and sensitive enough and was successfully applied to a pharmacokinetic study.     

Automated, fast and sensitive quantification of 17 alpha-hydroxy-progesterone, androstenedione and testosterone by tandem mass spectrometry with on-line extraction

Plasma 17 alpha-hydroxyprogesterone (17-OHP), androstenedione and testosterone measurements are important for the diagnosis and monitoring of hyperandrogenic disorders, most importantly for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. The reliability of immunoassays has proved questionable especially for newborns and children. In order to reduce the analytical interferences due to cross-reactivity or matrix effects, to improve accuracy and shorten the analysis time, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with atmospheric pressure chemical ionization (APCI) for simultaneous measurement. An on-line extraction cartridge with column-switching technique and liquid chromatography over a Chromolith RP 18 e column allow a rapid and easy quantification. The lowest limit of detection was 0.03-0.06 microg/L. Our method has proved linear up to 250 microg/L (r=0.999). Recoveries (S.D.) of 17-OHP, androstenedione and testosterone in plasma were 100% (5), 102% (2) and 92% (4), respectively. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for 17-OHP (excluding neonate samples) was y=1.942 x+0.255 nmol/L (r=0.695; n=97). In comparison to our values, the immunoassay generally overestimates steroid concentration. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for testosterone was y=0.963 x+0.035 nmol/L (r=0.955; n=107). Preliminary reference intervals for children were determined as a function of age and sex. The sensitivity and specificity of the LC-MS/MS method offer advantages over routine immunoassays due to the elimination of interferences especially for newborns, high throughput and short chromatographic run time.     

Determination of schizandrin in rat plasma by high-performance liquid chromatography-mass spectrometry and its application in rat pharmacokinetic studies

A sensitive liquid chromatography-mass spectrometric (LC/MS) method for the quantification of schizandrin in rat plasma was developed and validated after solid-phase extraction (SPE). Chromatographic separation was achieved on a reversed-phase Shimadzu C(18) column with the mobile phase of acetonitrile-sodium acetate (10 micromol/L) and step gradient elution resulted in a total run time of about 11.7 min. The analytes were detected using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (0.005-2.000 microg/mL) (r=0.9999). Lower limit of quantification (LLOQ) was 5 ng/mL and the lower limit of detection (LLOD) was 2 ng/mL using 100 microL plasma sample. Average recoveries ranged from 75.85 to 88.51% in plasma at the concentrations of 0.005, 0.100 and 1.000 microg/mL. Intra- and inter-day relative standard deviations were 5.95-12.93% and 3.87-14.53%, respectively. This method was successfully applied for the pharmacokinetic studies in rats.     

Simultaneous determination and confirmation of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscle by liquid chromatography-tandem mass spectrometry

A reliable and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) confirmation method has been developed for the simultaneous determination of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in chicken muscle. Samples were extracted with basic ethyl acetate, defatted with hexane, and cleaned up on Oasis MCX cartridges. LC separation was achieved on a XTerra C(18) column with gradient elution using a mobile phase composed of acetonitrile and water at a flow rate of 0.20 mL/min. The analysis was carried out on a triple-quadrupole tandem mass spectrometer in the multiple reaction monitoring (MRM) mode via electrospray interface operated in the positive and negative ionization modes, with deuterated chloramphenicol-d5 (d(5)-CAP) as the internal standard. The method validation was performed according to the criteria of Commission Decision 2002/657/EC. Four identification points were obtained for each analyte with one precursor ion and two product ions. Limits of detection (LODs) were 0.1 microg/kg for CAP, 0.2 microg/kg for FF and 1 microg/kg for TAP and FFA in chicken muscle. Linear calibration curves were obtained over concentration ranges of 0.3-20 microg/kg for CAP, 0.5-20 microg/kg for FF and 3-100 microg/kg for TAP and FFA in tissues. Mean recoveries of the 4 analytes ranged from 95.1% to 107.3%, with the corresponding intra- and inter-day variation (relative standard deviation, R.S.D.) less than 10.9% and 10.6%, respectively. The decision limit (CCalpha) and detection capability (CCbeta) of the method were also reported.     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

Determination of huperzine A in formulated products by reversed-phase-liquid chromatography using diode array and electrospray ionization mass spectrometric detection.

A precise and selective high-performance liquid chromatographic (HPLC) method with diode-array detection for quantifying huperzine A in formulated products was developed and validated. A liquid chromatographic-mass spectrometric (LC/MS) procedure was devised to confirm the HPLC method. Huperzine A was dissolved in 1,2-dichloroethane, chromatographed on a YMCBasic C18 column, and detected at 308 nm. A gradient mobile phase of 10 mM ammonium acetate (pH = 3.5)--methanol was used. Identification was based on retention time, UV spectra and mass spectra by comparison with a commercial standard. The UV peak areas were used for quantitation of huperzine A content. The correlation coefficient (R2) of the calibration curve was 1 over the range 0.8-11.6 microg/ml. Overall recovery of huperzine A was 103.9% +/- 1.8 (mean +/- SD). Relative standard deviations for intra- and interday precision were < 2%.     

Quantitative determination of besifloxacin, a novel fluoroquinolone antimicrobial agent, in human tears by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method was developed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the quantification of besifloxacin in human tears using sparfloxacin as the internal standard (IS). Besifloxacin was extracted from human tear samples using an ammonium formate buffer at pH 3.25. The method was validated over a concentration range of 2-2000 ng/mL, with a total run time of less than 4 min. The overall intra- and inter-day precision for this method was less than 6%. The method was used to measure besifloxacin concentrations in tear samples collected after topical ocular administration to humans; besifloxacin concentrations were 610+/-540 microg/g (15 min) and 1.60+/-2.28 microg/g (24h).     

Novel generic UPLC/MS/MS method for high throughput analysis applied to permeability assessment in early Drug Discovery

A novel generic ultra performance liquid chromatography-tandem mass spectrometric (UPLC/MS/MS) method for the high throughput quantification of samples generated during permeability assessment (PAMPA) has been developed and validated. The novel UPLC/MS/MS methodology consists of two stages. Firstly, running a 1.5min isocratic method, compound-specific multiple reaction monitoring (MRM) methods were automatically prepared. In a second stage, samples were analyzed by a 1.5min generic gradient UPLC method on a BEH C18 column (50mmx2.1mm). Compounds were detected with a Waters Micromass Quattro Premier mass spectrometer operating in positive electrospray ionization using the compound-specific MRM methods. The linearity for the validation compounds (caffeine, propranolol, ampicillin, atenolol, griseofulvin and carbamazepine) typically ranges from 3.05nM to 12,500nM and the limits of detection for all generically developed methods are in the range between 0.61nM and 12nM in an aqueous buffer. The novel generic methodology was successfully introduced within early Drug Discovery and resulted in a four-fold increase of throughput as well as a significant increase in sensitivity compared to other in-house generic LC/MS methods.     

Determination of oxytetracycline levels in rainbow trout serum on a biphenyl column using high-performance liquid chromatography

We developed a simple and sensitive high-performance liquid chromatography method on a biphenyl column to determine oxytetracycline (OTC) levels in rainbow trout serum. The assay used deproteination, filtration, and subsequent separation on a reverse-phase biphenyl column, with UV detection at 355 nm. OTC (7.8-7.9 min) was completely resolved from structurally similar riboflavin (10.4-10.5 min), a common feed supplement. Estimated limits of detection and quantitation of OTC were 0.01 and 0.04 microg/mL, respectively. The average recovery for OTC was 102% with a R.S.D. of 8.34%. Calibration standards were linear from 0.01 to 10 microg/mL.