Characterization of forced degradation products of ketorolac tromethamine using LC/ESI/Q/TOF/MS/MS and in silico toxicity prediction

Ketorolac, a nonsteroidal anti‐inflammatory drug, was subjected to forced degradation studies as per International Conference on Harmonization guidelines. A simple, rapid, precise, and accurate high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (LC/ESI/Q/TOF/MS/MS) method has been developed for the identification and structural characterization of stressed degradation products of ketorolac. The drug was found to degrade in hydrolytic (acidic, basic, and neutral), photolytic (acidic, basic, and neutral solution), and thermal conditions, whereas the solid form of the drug was found to be stable under photolytic conditions. The method has shown adequate separation of ketorolac tromethamine and its degradation products on a Grace Smart C‐18 (250 mm × 4.6 mm i.d., 5 µm) column using 20 mM ammonium formate (pH = 3.2): acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0 ml/min. A total of nine degradation products were identified and characterized by LC/ESI/MS/MS. The most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation of the [M + H]⁺ ions of ketorolac and its degradation products. In silico toxicity of the drug and degradation products was investigated by using topkat and derek softwares. The method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonization guidelines. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of forced degradation products of pazopanib hydrochloride by UHPLC‐Q‐TOF/MS and in silico toxicity prediction

Pazopanib (PZ), an anti‐cancer drug, was subjected to forced degradation under hydrolytic (acid, base and neutral), oxidative, photolytic and thermal stress conditions as per International Conference on Harmonization guidelines. A selective stability indicating validated method was developed using a Waters Acquity UPLC HSS T3 (100 × 2.1 mm, 1.7 µm) column in gradient mode with ammonium acetate buffer (10 m m , pH 5.0) and acetonitrile. PZ was found to degrade only in photolytic conditions to produce six transformation products (TPs). All the TPs were identified and characterized by liquid chromatography/atmospheric pressure chemical ionization–quadrupole‐time of flight mass spectrometry experiments in combination with accurate mass measurements. Plausible mechanisms have been proposed for the formation of TPs. In silico toxicity was predicted using TOPKAT and DEREK softwares for all the TPs. The TP, N4‐(2,3‐dimethyl‐2H‐indazol‐6‐yl)‐N4‐methylpyrimidine‐2,4‐diamine, was found to be genotoxic, whereas all other TPs with sulfonamide moiety were hepatotoxic. The data reported here are expected to be of significance as this study foresees the formation of one potential genotoxic and five hepatotoxic degradation/transformation products. Copyright © 2015 John Wiley & Sons, Ltd.     

LC-MS/TOF, LC-MSn, on-line H/D exchange and LC-NMR studies on rosuvastatin degradation and in silico determination of toxicity of its degradation products: a comprehensive approach during drug development

The present study dealt with the forced degradation behaviour of rosuvastatin under ICH prescribed stress conditions. The drug was found to be labile under acid hydrolytic and photolytic conditions, while it was stable to base/neutral hydrolytic, oxidative and thermal stress. In total, 11 degradation products were formed, which were separated on a C-18 column using a stability-indicating method. LC-MS analyses indicated that five degradation products had the same molecular mass as that of the drug, while the remaining six had 18 Da less than the drug. Structure elucidation of all the degradation products was executed using sophisticated and modern structural characterization tools, viz. LC-MS/TOF, LC-MSⁿ, on-line H/D exchange and LC-NMR. The degradation pathway and mechanisms of degradation of the drug were delineated. Additionally, in silico toxicity was predicted for all the degradation products using TOPKAT and DEREK software and compared with the drug. This study demonstrates a comprehensive approach of degradation studies during the drug development phase.

Ultra-trace determination of aquaculture chemotherapeutants and degradation products in environmental matrices by LC-MS/MS

Two of the most common products currently used to control parasitic sea lice in fin fish aquaculture, salmon in particular, are Slice® and AlphaMax®. Emamectin benzoate (EB) is the active ingredient in Slice® and deltamethrin is the active ingredient in AlphaMax®. Several analytical methods have been developed for the determination of the active ingredients in these products but these have been focused on specific matrices and lack the sensitivity and versatility required in environmental monitoring. Here we present a validated, versatile, and simple analytical method for the determination of EB, its desmethyl degradation product (AB), and deltamethrin in a wide range of environmental matrices (sea water, marine sediment, and tissue). Sediments and tissues were extracted by accelerated solvent extraction (ASE®) and sample cleanup was achieved by solid phase extraction (SPE) while sea water was extracted using SPE disks. Analyte identification and quantification was based on liquid chromatography tandem mass spectrometry (LC-MS/MS) instrumentation with electrospray ionization, and multiple reaction monitoring (MRM). Method detection limits for the target analytes was in the parts per trillion (pg?g^?1) level for tissue and sediment and parts per quadrillion (pg?L^?1) for water. Except for deltamethrin in sea water, method performance in terms of analyte recoveries was better than 60%, and the method precision was RSD<20%. The method was used to determine EB and AB concentrations in water, sediment and prawn tissue samples collected near salmon aquaculture sites treated with Slice®. A distinct concentration gradient was observed in the immediate vicinity (within 50 to 100?m radius) of the salmon aquaculture sites where EB was detected at low ng?g^?1 levels for tissue (EB ranged from 0.041 to 3.0?ng?g^?1) and sediment (EB ranged from 0.051 to 35?ng?g^?1) and pg?L^?1 levels (EB ranged from 3 to 209?pg?L^?1) for water samples.     

LC–MS/MS method for the characterization of the forced degradation products of Entecavir

A rapid, specific, and reliable isocratic LC–MS/MS method has been developed and validated for the identification and characterization of the stressed degradation products of Entecavir (ETV). ETV, an antiviral drug, was subjected to hydrolysis (acidic, alkaline, and neutral), oxidation, photolysis and thermal stress, as per the international conference on harmonization specified conditions. The drug showed extensive degradation under oxidative and acid hydrolysis stress conditions. However, it was stable to thermal, acidic, neutral, and photolysis stress conditions. A total of five degradation products were observed and the chromatographic separation of the drug and its degradation products were achieved on a Waters Symmetry C₁₈ (250 mm × 4.6 mm, id, 5 μm) column using 20 mM ammonium acetate (pH 3)/acetonitrile (50:50, v/v) as a mobile phase. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. The LC–MS method was validated with respect to specificity, linearity, accuracy, and precision. No previous reports were found in the literature regarding the degradation behavior of ETV.     

Determination of IGF-1 and IGF-2, their degradation products and synthetic analogues in urine by LC-MS/MS

Peptide analysis in doping controls by means of nano-UPLC coupled high resolution/high mass accuracy mass spectrometry provides the state-of-the-art technique in modern sports drug testing. The present study describes a recent application of this technique for the qualitative determination of different urinary insulin-like growth factor (IGF) related peptides. After simultaneous isolation by solid phase extraction and magnetic particle-based immunoaffinity purification, target analytes (IGF-1, IGF-2, Des1-3-IGF-1, R(3)-IGF-1 and longR(3)-IGF-1) were separated by nano-liquid chromatography prior to mass spectrometric detection. Endogenously produced IGF-1 and IGF-2, as well as the degradation product Des1-3-IGF-1, were frequently detected in urine samples from healthy volunteers in a concentration range of 20-400 pg mL(-1). The impact of IGF binding proteins (IGFBPs), being also present in urine, was potentially estimated by an additional ultrafiltration step in the sample preparation procedure. The synthetic analogue longR(3)-IGF-1, which is assumed to be subject to misuse by cheating athletes, was also analysed and detected in fortified urine samples. Besides the intact molecule, an N-terminally truncated degradation product Des1-10-longR(3)-IGF-1 was identified as the more stable target for doping controls using urine samples. The method was validated for qualitative purposes considering the parameters specificity, limit of detection (20-50 pg mL(-1)), recovery (10-35%), precision (<20%), linearity, robustness and stability.     

Liquid chromatography/mass spectrometric studies on atorvastatin and its stress degradation products

A comprehensive mass fragmentation pathway of atorvastatin, which has not been reported so far, was established by subjecting the drug to multi-stage mass spectrometric (MSn) studies. It was used along with liquid chromatography/mass spectrometric (LC/MS) and liquid chromatography/time-of-flight mass spectrometric (LC/TOFMS) analyses to identify the drug degradation products formed under stress conditions of hydrolysis, oxidation and photolysis. Other than lactone, which is a reported hydrolysis product, six unknown hydrolytic products could be identified, viz., dehydrated drug, dehydrated drug lactone, and diastereomers of the drug, drug lactone, dehydrated drug, and dehydrated drug lactone. Among the two products separated under oxidative conditions, one was lactone, again formed as a result of drug hydrolysis in an acidic environment of peroxide solution. The other was similar to a reported oxidative product. Under photolytic conditions in solution, one new product could be identified, while most of the others matched with those known from the literature. Hence overall a more complete degradation pathway of the drug was established than known at present, by using a stress testing approach and employing LC/MS techniques.     

Determination of bifonazole and identification of its photocatalytic degradation products using UPLC‐MS/MS

The main goal of the presented work was to investigate the effect of ZnO or/and TiO₂ on the stability of bifonazole in solutions under UVA irradiation. To this end, a simple and reproducible UPLC method for the determination of bifonazole in the presence of its photocatalytic degradation products was developed. Linearity was studied in the range of 0.0046–0.15 mg mL⁻¹ with a determination coefficient of 0.9996. Bifonazole underwent a photocatalytic degradation process under the experimental conditions used. Comparative studies showed that combination of TiO₂/ZnO (1:1 w /w) was a more effective catalyst than TiO₂ or ZnO with a degradation rate of up to 67.57% after 24 h of irradiation. Further, kinetic analyses indicated that the photocatalytic degradation of bifonazole in the mixture of TiO₂/ZnO can be described by a pseudo‐first order reaction. Statistical comparison clearly indicated that the presence of TiO₂/ZnO also affected the stability of bifonazole from a cream preparation after 15 h of UVA exposure (p < 0.05). Ten photodegradation products of bifonazole were identified for the first time and their plausible fragmentation pathways, derived from MS/MS data, were proposed. The main pathway in the photocatalytic transformation of bifonazole in the presence of ZnO or/and TiO₂ involves hydroxylation of the methanetriyl group and/or adjacent phenyl rings and cleavage of the imidazole moiety.     

A validated LC-MS/MS method for the determination of vinflunine in plasma and its application to pharmacokinetic studies

A rapid, simple and sensitive LC‐MS/MS method for the quantification of vinflunine in plasma was developed and validated. The analysis involved a simple liquid–liquid extraction. After making alkaline with NaOH, plasma was extracted with methyl tert‐butyl ether and the organic extract was then evaporated and the residue was reconstituted in mobile phase. The reconstituted solution was injected into an HPLC system and was subjected to reverse‐phase HPLC on a 5 µm ODS‐3 column at a flow‐rate of 0.2 mL/min. The mobile phase consisted of ammonium acetate (0.02 mol/L, pH = 3.0) and acetonitrile (20:80). Vinflunine was detected in the single ion monitoring mode using target ions at m/z 817.4/160.1/142.3 for vinflunine and m/z 447.2/128.3/112.1 for gefitinib (internal standard). Standard curves were linear over the concentration range of 5–1000 ng/mL. The mean predicted concentrations of the quality control samples deviated by less than 2% from the corresponding nominal values; the intra‐assay and inter‐assay precisions of the assay were within 7% relative standard deviation. The extraction recovery of vinflunine was more than 80%. The validated assay was applied to a pharmacokinetic study of vinflunine in plasma following the administration of a single vinflunine injection (2 mg/kg). Copyright © 2011 John Wiley & Sons, Ltd.     

New forced degradation products of vildagliptin: Identification and structural elucidation using LC-MS,with proposed formation mechanisms

Abstract

A new and simple RP-HPLC-UV method was developed for well-separation of vildagliptin raw material and its degradation products at different conditions; it uses of ammonium acetate buffer at pH= 7.5 and methanol with Athena C18 -WP (250?mm) column. Results show that six degradants have been identified using LC–MS technique, in addition to the NMR approach in some cases. One degradant at relative retention time (RRT) 1.3 was formed under acidic condition and designated as 2-((1R, 3S, 5R, 7S)-3-hydroxyadamantan-1-yl) hexahydropyrrolo[1,2-a]pyrazine-1,4-dione at m/z = 304. Three degradants were formed under various conditions of basic hydrolysis at RRTs 1.2, 0.6 and 0.4 with following names and molar masses (m/z), respectively: 1-(((1S, 3S, 5S, 7S)-1,3-dihydroxyadamantan-2-yl)glycyl)pyrrolidine-2-carboxamide at m/z = 337.2, 1-(((1R, 3S, 5R, 7S)-3-hydroxyadamantan-1-yl)glycyl)pyrrolidine-2-carboxamide at m/z = 321.1 and (1,4-dioxo-1,4,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazin-3-yl)glycylproline at m/z = 322.6. Another three degradants were also formed under oxidative oxidations of vildagliptin, one at RRT 0.38 and designated as N-hydroxy-N-((1R, 3S, 5R, 7S)-3-hydroxyadamantan-1-yl) glycinate with m/z 241.1, the second one was identical to that formed under basic hydrolysis at RRT 0.6 and the last one has RRT 0.8 and was identified as (1S, 3R, 5R, 7S)-3-(hydroxyamino)adamantan-1-ol at m/z 183.1. Formation mechanisms for the degradation products were described.     

Simultaneous determination of 32 antibiotics in aquaculture products using LC-MS/MS

An analytical multiclass, multi-residue method for the determination of antibiotics in aquaculture products was developed and validated. A fast, cheap, and straightforward extraction procedure followed by liquid chromatography-tandem mass spectrometry analysis was proposed. This method covers 32 antibiotics of different classes, which are frequently used in aquaculture. Three different extraction procedures were compared, and the extraction with acetonitrile (0.1 vol. % formic acid) showed the best results. The selected extraction procedure was validated at four different fortification levels (10 μg kg^?1, 25 μg kg^?1, 50 μg kg^?1, and 100 μg kg^?1). Recoveries of the tested antibiotics ranged from 70 % to 120 %, with the relative standard deviation (RSD) of triplicates lower than 20 %. The limits of quantification (LOQ) ranged from 0.062 μg kg^?1 to 4.6 μg kg^?1, allowing for the analysis of trace levels of these antibiotics in aquaculture products. The method was applied to the analysis of selected antibiotics in fish and shrimp meat available in the Czech market.     

LC/MS/MS identification of some folic acid degradation products after E-beam irradiation

Folates belong to the B vitamin group based on the parental compound folic acid (FA). They are involved in important biochemical processes like DNA synthesis and repair. FA is composed of a pteridine ring, p-aminobenzoic acid and glutamate moieties. The human metabolism is not able to synthesize folates and therefore obtain them from diet. FA, a synthetic vitamin, is used as a food fortificant because of its low price, relative stability and increased bioavailability compared to natural folate forms. FA is known to be a sensitive compound easily degradable in aqueous solution by ultraviolet and visible light towards various by-products. Irradiation is a process for preservation of foods that uses accelerated electrons, gamma rays or X-rays. Irradiation is proposed for the treatment of various food products, eliminating or reducing pathogens and insects, increasing the storage time and replacing chemical fumigants. This study concerns the identification of degradation products of FA after E-beam irradiation. FA aqueous solutions were irradiated with a Van de Graaff electrons beam accelerator (2 MeV, 100 μA current, 20 cm scan width, dose rate about 2 kGy/s). Applied doses were between 0 (control) and 10.0 kGy. Absorbed doses were monitored with FWT 60.00 radiochromic dosimeters.     

Characterization of different poly(2‐oxazoline) block copolymers by MALDI‐TOF MS/MS and ESI‐Q‐TOF MS/MS

A complete library of poly(2‐oxazoline) block copolymers was synthesized via cationic ring opening polymerization for the characterization by two different soft ionization techniques, namely matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF MS). In addition, a detailed characterization was performed by tandem MS to gain more structural information about the block copolymer composition and its fragmentation behavior. The fragmentation of the poly(2‐oxazoline) block copolymers revealed the desired polymer structure and possible side reactions, which could be explained by different mechanisms, like 1,4‐ethylene or hydrogen elimination and the McLafferty +1 rearrangement. Polymers with aryl side groups showed less fragmentation due to their higher stability compared to polymers with alkyl side groups. These insights represent a further step toward the construction of a library with fragments and their fragmentation pathways for synthetic polymers, following the successful examples in proteomics. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

One single LC-MS/MS analysis for both phenolic components and tanshinones in Radix Salviae Miltiorrihizae and its medicinal products

A LC-MS/MS method was developed for the separation and simultaneous determination of phenolic components including danshensu, protocatechuic acid, protocatechuic aldehyde and caffeic acid as well as tanshinones including cryptotanshinone, tanshinone I and tanshinone IIA in samples of Radix Salviae Miltiorrhizae and Salviae Miltiorrhizae tablet. Triple quadrupole mass spectrometry was optimized in both positive and negative ion multiple reaction monitoring modes for the simultaneous quantitative analysis of the two different types of active components by using a time-segment program. The method gave recoveries of 85.4-106.4% with relative standard deviations of 2.4-8.0% for the spiked herb samples. The limits of detection were 0.30-0.83 μg/g for the analysis of 1.0 g Radix Salviae Miltiorrhizae or tablet samples.     

液相色谱-串联质谱法快速检测番茄制品中苏丹红

采用HPLC/MS-MS鉴定和检测番茄制品中苏丹红染料Ⅰ～Ⅳ。番茄制品中苏丹红Ⅰ、Ⅱ、Ⅲ、Ⅳ,经正己烷加乙酸溶液提取,氧化铝柱净化,再用二氯甲烷洗脱苏丹红,经浓缩至干,流动相定容,液相色谱串联质谱测定,外标法定量。将已用HPLC法测定过的10个可疑样品,用该方法进行检测,结果有4个检了苏丹红Ⅰ。采用本方法检测苏丹红Ⅰ～Ⅳ,检测限均可达到1.0μg/kg,在添加浓度1～100μg/kg之间线性关系良好,相关系数(r)均>0.9990,对于1、5、10μg/kg水平添加回收率在72.3%～103%之间,相对标准偏差在2.5%～9.8%之间。     

Characterization of the biotransformation pathways of clomiphene, tamoxifen and toremifene as assessed by LC-MS/(MS) following in vitro and excretion studies

The use of selective oestrogen receptor modulators has been prohibited since 2005 by the World Anti-Doping Agency regulations. As they are extensively cleared by hepatic and intestinal metabolism via oxidative and conjugating enzymes, a complete investigation of their biotransformation pathways and kinetics of excretion is essential for the anti-doping laboratories to select the right marker(s) of misuse. This work was designed to characterize the chemical reactions and the metabolizing enzymes involved in the metabolic routes of clomiphene, tamoxifen and toremifene. To determine the biotransformation pathways of the substrates under investigation, urine samples were collected from six subjects (three females and three males) after oral administration of 50 mg of clomiphene citrate or 40 mg of tamoxifen or 60 mg of toremifene, whereas the metabolizing enzymes were characterized in vitro, using expressed cytochrome P450s and uridine diphosphoglucuronosyltransferases. The separation, identification and determination of the compounds formed in the in vivo and in vitro experiments were carried out by liquid chromatography coupled with mass spectrometry techniques using different acquisition modes. Clomiphene, tamoxifen and toremifene were biotransformed to 22, 23 and 18 metabolites respectively, these phase I reactions being catalyzed mainly by CYP3A4 and CYP2D6 isoforms and, to a lesser degree, by CYP3A5, CYP2B6, CYP2C9, CYP2C19 isoforms. The phase I metabolic reactions include hydroxylation in different positions, N-oxidation, dehalogenation, carboxylation, hydrogenation, methoxylation, N-dealkylation and combinations of them. In turn, most of the phase I metabolites underwent conjugation reaction to form the corresponding glucuro-conjugated mainly by UGT1A1, UGT1A3, UGT1A4, UGT2B7, UGT2B15 and UGT2B17 isoenzymes.     

Identification of photocatalytic degradation products of diazinon in TiO2 aqueous suspensions using GC/MS/MS and LC/MS with quadrupole time-of-flight mass spectrometry

The photocatalytic degradation of the organophosphorus insecticide diazinon in aqueous suspensions has been studied by using titanium dioxide as a photocatalyst. The degradation of the insecticide was a fast process and included the formation of several intermediates that were identified using GC/ion-trap mass spectrometry with EI or CI in positive and negative ionization mode and HPLC/electrospray-QqTOF mass spectrometry. Since primarily hydroxy derivatives were identified in these aqueous suspensions, the mechanism of degradation was probably based on hydroxyl radical attack. The initial oxidative pathways of the degradation of diazinon involved the substitution of sulfur by oxygen on the Pz.dbnd6;S bond, cleavage of the pyrimidine ester bond, and oxidation of the isopropyl group. Exact mass measurements of the derivatives allowed the elemental formula of the molecules to be determined confidently. Similarities to the metabolic pathways occurring in living organisms were observed.     

LC and LC-MS study on establishment of degradation pathway of glipizide under forced decomposition conditions

Forced degradation studies on glipizide are conducted under the conditions of hydrolysis, oxidation, photolysis, and dry heat. The solutions are subjected to liquid chromatographic (LC) investigations to establish the number of products formed in each condition. The degradation products are characterized through isolation and subsequent NMR, IR, and MS spectral analyses, or through LC-mass spectrometry (MS) fragmentation pattern study. The drug is shown to degrade in 0.1M HCl at 85 degrees C to two products: 5-methyl-N-[2-(4-sulphamoylphenyl)ethyl]pyrazine-2-carboxamide (II) and methyl N-[4-[2-{(5-methyl-2-pyrazinoyl)amino}ethyl] phenyl]sulfonyl carbamate (III). The latter, a methyl ester, is formed only in the presence of methanol (used as a solubilizer), and does not appear on use of acetonitrile. III is shown to convert to II on continued heating in acid. The drug degrades slowly in water at the same temperature, and both II and III could be seen in the chromatograms until the end of the study. The heating of the drug in alkali (0.1M NaOH) at 85 degrees C yields 5-methyl-2-pyrazinecarboxylic acid (IV), along with a small quantity of 4-(2-aminoethyl) benzenesulfonamide (I). On extended heating in the same condition, a new product, 4-(2-aminoethyl)-N,N-bis[(cyclohexylamino)carbonyl] benzenesulfonamide (VI) is formed in small quantities. At the lower temperature of 40 degrees C, the drug converts under each hydrolytic condition and in both the absence and presence of light to products II, III, or IV, along with a new product, 1-cyclohexyl-3-[[4-(2aminoethyl)phenyl] sulfonyl]urea (V). The light catalyzes formation of V, and it is formed until one or two weeks, after which its level decreases. The drug remains stable in 30% H2O2, except that products II and III appear as small peaks due to acidic character of the peroxide solution. Also, the drug remains unaffected in solid state under thermal and photolytic stress conditions. Based on the results, a more complete picture on degradation pathway of the drug is obtained, highlighting a clear advantage of the approach suggested by International Conference on Harmonization.     

Bioanalytical method development and validation of novel antithrombotic agent S002-333 by LC-MS/MS and its application to pharmacokinetic studies

A rapid, sensitive and simple liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method using an electrospray ionization (ECI) source for the quantification of novel anti‐thrombotic agent S002‐333 [2‐(4‐methoxy‐benzenesulfonyl)‐2,3,4,9‐tetrahydro‐1H‐β‐carboxylic acid amide] in rabbit plasma was developed and validated. The extraction from plasma was carried out by simple protein precipitation extraction method. The chromatographic separation was performed on an Ultramex Cyno, (150 × 4.6 mm, 5 µm) with a guard column, using acetonitrile–water (75:25,v/v) with flow rate of 0.6 mL/min as the mobile phase. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z transitions 386.4/215.4 for S002‐333 and m/z 393.4/171for the internal standard dexamethasone, using positive ion mode. The MS/MS response was linear over the concentration range from 1.56 to 200 ng/mL, with a lower limit of detection of 0.78 ng/mL. The accuracy and precision of the method were within the acceptable limit of ±20% at the lower limit of quantitation and ±15% at other concentrations and showed no significant matrix effect. The validated method can be used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic studies Copyright © 2010 John Wiley & Sons, Ltd.