A subnanogram API LC/MS/MS quantitation method for depsipeptide FR901228 and its preclinical pharmacokinetics

A highly sensitive and specific atmospheric pressure ionization (API) liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method for the quantitation of depsipeptide FR901228 (NSC-630176, FR), a naturally occurring antitumor agent, was developed and validated. FR was extracted from human or rat plasma along with the internal standard, t-Boc-Met-Leu-Phe (BMLP) with ethyl acetate. Components in the extract were separated on a 5-microm C8 Spherisorb 50 x 4.6 mm i.d. column by isocratic elution with methanol/acetonitrile/12 mM ammonium acetate (60:10:30, v/v/v). The liquid flow was passed through a presource splitter and 5% of the eluate was introduced into the API source. The components were analyzed in the multiple-reaction monitoring (MRM) mode to enhance specificity. Linear calibration curves were obtained in the range of 0.1-100.0 ng/ml with 0.5 ml human plasma and 0.5-100.0 ng/ml with 0.1 ml rat plasma. The limit of quantitation (LOQ) was 0.1 ng/ml using 0.5 ml human plasma and 0.5 ng/ml using 0.1 ml rat plasma. The overall within-day precision was below 12% in human plasma and below 7% in rat plasma; and the between-day precision was below 10.2% in human plasma and 7.2% in rat plasma. The accuracy at low, medium and high levels ranged from 99.3 to 111.7% in human plasma and 96.2-107.3% in rat plasma. The high sensitivity permitted pharmacokinetic study of FR in the rat at a single i.v. dose as low as 1 mg/kg. At this dose, plasma FR levels declined biexponentially with a mean terminal t(1/2) of 187.7 min (n = 6) and were detectable up to 24 h. After an oral dose at 5 mg/kg, plasma FR levels were highly erratic and yielded a mean bioavailability of 1.6% (n = 6). At a higher oral dose of 50 mg/kg, a mean bioavailability of 10.6% was obtained, both being estimated by a non-crossover method.     

Simultaneous determination of SU5416 and its phase I and phase II metabolites in rat and dog plasma by LC/MS/MS

SU5416, Z-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone, is a cytostatic substance in development as an anti-angiogenic agent. SU5416 has several phase I and phase II metabolites including SU9838, SU6595, SU6689, 5′-hydroxy glucuronide of SU5416 and 5′-acyl glucuronide of SU5416. In order to support the preclinical studies, a liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) method for simultaneous determination of SU5416 and its metabolites in rat and dog plasma was developed. This method is fast, simple, sensitive (LOQ=2.0 ng/ml), reproducible and has a wide linear range (2.0–5000 ng/ml for SU5416, 2.0–2000 ng/ml for SU6689 and 2.0–1000 ng/ml for SU9838 and SU6595). This method was applied to rat and dog plasma samples obtained from pharmacokinetic and toxicokinetic studies.     

A rapid and sensitive LC/MS/MS assay for quantitative determination of digoxin in rat plasma

Digoxin is a cardiac glycoside that is widely used for the treatment of congestive heart failure. To evaluate pharmacokinetics of digoxin in rats, a sensitive LC/MS/MS assay was developed and validated for the determination of digoxin concentration in rat plasma. For detection, a Sciex API3000 LC/MS/MS with atmospheric pressure ionization (API) mass spectrometry turbo ion spray inlet in the positive ion-multiple reaction monitoring mode was used to monitor precursor→product ions of m/z 798.6→651.6 for digoxin and m/z 577.6→433.3 for oleandrin, the internal standard (IS). The standard curve was linear (r²≥0.999) over the digoxin concentration range of 0.1–100 ng/ml in plasma for digoxin. The mean predicted concentrations of the quality control samples deviated by <5.8% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8.6% relative standard deviation. At the lower limit of quantitation (LLQ) of 0.1 ng/ml, the mean deviation of predicted concentrations from the nominal value was within 3.7%. The extraction recoveries of digoxin and internal standard were 82.7±3.9 and 105.9±2.3%, respectively. The present method was successfully applied to characterization of pharmacokinetic profiles of digoxin in rats after oral administration.     

HPLC-MS／MS法测定大鼠血浆中兰索拉唑及其代谢产物的浓度

目的：建立大鼠血浆中兰索拉唑及其代谢产物5-羟基兰索拉唑、兰索拉唑砜的HPLC-MS／MS测定方法。方法：色谱条件：色谱柱：Diamonsil C18柱（150mm×2．1mm,5um）;流动相：乙腈-水（合0．01％甲酸及2mmol·L-1的醋酸铵（43：57,V／V）;流速：0．3ml·min-1;柱温：40℃;进样量10ul。质谱条件：电喷雾离子源（ESI）,以多反应监测离子方式测定兰索拉唑及其代谢产物,选择性监测质荷比（m／z）为368．0／163．9（兰索拉唑）,384．1／179．9（5-羟基兰索拉唑）,383．9／115．9（兰索拉唑砜）,326．0／280．1（内标奥美拉唑）。样品用乙腈沉淀蛋白处理。结果：兰索拉唑、5-羟基兰索拉唑、兰索拉唑砜的线性范围分别为11．40～4560．00,1．26～504．00,1．24～496．00ng·ml-1;定量下限分别为11．40,1．26,1．24ng·ml-1;批内、批间精密度RSD均〈15％。结论：该方法灵敏、准确、快速、专属性好,适用于兰索拉唑及其代谢产物在大鼠体内的药代动力学研究。     

Liquid chromatography/tandem mass spectrometry for the determination of carbamazepine and its main metabolite in rat plasma utilizing an automated blood sampling system

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of carbamazepine and its main metabolite carbamazepine 10,11-epoxide in rat plasma is described. The method consists of a liquid-liquid extraction procedure and electrospray LC/MS/MS analysis. The chromatographic separation was achieved within 5 min using a C(8) (150 mm x 2.1mm) 5 microm column with a mobile phase composed of water/acetonitrile/acetic acid (69.5:30:0.5, v/v/v) at a flow rate of 0.4 ml/min. D(10)-carbamazepine is used as the internal standard for all compounds. Analytes were determined by electrospray ionization tandem mass spectrometry in the positive ion mode using selected reaction monitoring (SRM). Carbamazepine was monitored by scanning m/z 237-->194, carbamazepine 10,11-epoxide by m/z 253-->210 and d(10)-carbamazepine by m/z 247-->204. The lower limit of quantitation (LLOQ) is 5 ng/ml for each analyte, based on 0.1 ml aliquots of rat plasma. The extraction recovery of analytes from rat plasma was over 87%. Intra-day and inter-day assay coefficients of variations were in the range of 2.6-9.5 and 4.0-9.6%, respectively. Linearity is observed over the range of 5-2000 ng/ml. This method was used for pharmacokinetic studies of carbamazepine and carbamazepine 10,11-epoxide in response to two different blood sampling techniques (i.e., manual sampling versus automated sampling) in the rat. Several differences between the two sampling techniques suggest that the method of blood collection needs to be considered in the evaluation of pharmacokinetic data.     

LC/MS/MS analysis of vesnarinone and its principal metabolites in plasma and urine

An LC/MS/MS assay was developed and successfully used to quantitate vesnarinone and its principal metabolites (OPC-8230, OPC-18136, and OPC-18137) in human plasma and urine. Samples were pre-treated with liquid–solid extraction followed by simultaneous monitoring of primary and daughter ions which were used for the identification and quantitation of the analytes on LC/MS/MS. This assay offers advantages of specificity, speed and greater sensitivity over the previously developed HPLC-UV assay. The lower limit of quantitation is 500 ng ml⁻¹ for vesnarinone and 20 ng ml⁻¹ for OPC-8230, OPC-18137, and OPC-18136 in plasma. Methodology is similar for the estimation of these analytes in urine with the lower limit of quantitation being 500 ng ml⁻¹ for vesnarinone and 100 ng ml⁻¹ for each metabolite. Ascorbic acid was added to stabilize the analytes from degradation. This LC/MS/MS method was developed to overcome many practical problems associated with the HPLC method. The LC/MS/MS method offers the flexibility of analyzing additional metabolites and changing the linearity range to accommodate the differences in linear range (200–10 000 ng ml⁻¹ for vesnarinone and 20–1000 for metabolites) for the analytes.     

Specific,sensitive and accurate LC-MS/MS method for the measurement of levovirin in rat and monkey plasma

Levovirin is a guanosine nucleoside analogue and the L-enantiomer of ribavirin. Levovirin has a better safety profile than ribavirin, exerts similar immunomodulatory effects in a mouse efficacy model, and may provide a better therapeutic option than ribavirin in patients with chronic hepatitis C virus (HCV) infection. To facilitate pharmacokinetic studies, a LC-MS/MS method for the analysis of levovirin in rat and monkey plasma was developed and validated. The method involved adding ICN 10537 as an internal standard, protein precipitation with acetonitrile followed by separation on an Intersil Silica column, and quantification by a MS/MS system equipped with positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MS/MS reaction was selected to monitor the 245-->113 and 259-->128 transitions for levovirin and internal standard, respectively. The calibration curve was linear over a concentration range of 10-5000 ng/ml. The limit of quantitation was 10 ng/ml, the coefficient of variation (CV) was 3-5%, and the bias was 3-6%. Intra- and inter-day analysis of QC samples at 30, 1500 and 3500 ng/ml indicated that the method was precise (CV<6%) and accurate (bias <9%). Levovirin in rat and monkey plasma was stable at 5 degrees C for at least 24 h, 0 degrees C for at least 4 h, and after three freeze-thaw cycles. This specific, accurate and precise assay is useful in the study the pharmacokinetic characteristics of this compound.     

Pharmacokinetic study of the novel, synthetic trioxane antimalarial compound 97-78 in rats using an LC-MS/MS method for quantification

The present study has been designed to investigate the pharmacokinetic parameters of the novel trioxane antimalarial 97-78 (US Patent 6316493 B1, 2001) in male and female rats after single oral and intravenous administration. The pharmacokinetic profile of 97-78 was investigated in the form of its completely converted metabolite 97-63 after dose administration. Quantification of metabolite 97-63 in rat plasma was achieved using a simple and rapid LC-MS/MS method. The LC-MS/MS method has been validated in terms of accuracy, precision, sensitivity and recovery for metabolite 97-63 in rat plasma. The intra- and interday accuracy (% bias) and precision (% RSD) values of the assay were less than 10% for metabolite 97-63. The chromatographic run time was 4.0 min and the weighted (1/x2) calibration curves were linear over the range 1.56-200 ng/ml. This method was successfully applied for analysis of pharmacokinetic study samples. Maximum plasma concentrations of 97-63 at 47 mg/kg oral administration in male and female rats were 1986.6 ng/ml and 4086.7 ng/ml at time (Tmax) 0.92 h and 0.58 h, respectively. The area under the curve (AUC(0-infinity)), elimination half-life (t(1/2) beta) and mean residence time (MRT) were 4669.98 ng x h/ml, 2.8 h and 4.2 h in male and 11786.0 ng x h/ml, 4.52 h and 4.32 h in female rats respectively. After single oral and intravenous administration of 97-78 to male and female rats significant differences were observed in pharmacokinetic parameters (AUC and t (1/2) beta) for metabolite 97-63.     

液相色谱-串联质谱法测定人血浆中盐酸舍曲林浓度

目的：建立测定人血浆中盐酸舍曲林浓度的液相色谱-串联质谱法。方法：以替米沙坦为内标,内标法定量。流动相：乙腈-10mmol·L^-1乙酸铵-1%甲酸（70：30：0.1）;质谱采用离子喷雾离子化源,扫描方式为多重反应监测（MRM）,用于定量分析的离子反应分别为m/z306.3→m/z159.1（舍曲林）和m/z515.2→m/z276.1（替米沙坦）。结果：舍曲林和替米沙坦的保留时间分别为2.22min和2.44min;舍曲林的线性范围为0.5～50.0ng·mL^-1,r=0.9992,回归方程：Y=0.0021＋0.0217X,最低检测浓度为0.5ng·mL^-1;提取回收率在82%～90%范围内;日内相对标准差〈4%,日间精密度〈13%。结论：此法适合人体血浆盐酸舍曲林浓度的监测及生物利用度研究,结果准确、可靠。     

Sensitive and robust UPLC–MS/MS method to determine the gender-dependent pharmacokinetics in rats of emodin and its glucuronide

In this study, a sensitive and robust ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed, validated, and applied to determine gender-dependent pharmacokinetics of total emodin (aglycone + glucuronide) in male and female Sprague–Dawley rats. The lower limit of quantification for emodin and emodin glucuronide in rat plasma was 39 and 78 ng/ml, with signal-to-noise ratio of ≥10. Precision and accuracy studies showed emodin and emodin glucuronide plasma concentrations well within the 10% range in all studies. Plasma recovery of emodin and emodin glucuronide was always above 86% for low (emodin: 39 ng/ml; glucuronide: 78 ng/ml), 92% for medium (625 ng/ml), and 97% for high (10 000 ng/ml) concentrations. Furthermore, emodin showed more than 95% plasma stability under short-term and long-term storage conditions, as well as after three freeze–thaw cycles in the experiments. The developed and validated analytical method was successfully applied to study the gender-dependent 10-fold higher oral bioavailability of total emodin in male than female rats. The oral bioavailability of emodin and emodin glucuronide was also measured separately and showed a statistically significant gender difference in oral bioavailability of emodin and emodin glucuronide in rats.     

LC/MS/MS plasma assay for the peptidomimetic VLA4 antagonist I and its major active metabolite II: for treatment of asthma by inhalation.

In vitro and in animals, I is a potent and specific peptidomimetic for the potential treatment of airway inflammation in the pathogenesis of asthma. Preclinical studies indicated extensive conversion of I to an active metabolite II, and thus, a very sensitive assay for I and II was needed to support an inhalation ascending-dose study in man. The LC/MS/MS plasma/urine assay method (1.0 ml of sample) involves the following: liquid-liquid extraction of acidified plasma into pentane-ethyl acetate (90:10 v/v); evaporation of the organic extract, reconstitution into methanol; addition of water to the methanolic extract and freezing. After thawing, the extract is centrifuged and the clear supernatant injected for chromatography. Extract is chromatographed on a YMC ODS-AM column (50 x 2.0 mm). For detection, a Sciex 365 LC/MS/MS with an electrospray inlet and used in the positive ion, multiple reaction monitoring mode was used to monitor precursor-->fragment ions of m/z 709-->594 for I and m/z 513-->380 for II. The plasma assay was linear over the concentration range of 0.1-100 ng/ml in plasma for I and II. Accuracy and precision for I ranged from 97.9 to 102.1% of nominal with a 0.84-10.65% CV; similarly for II, 98.0-101.7% and 1.39-9.28% CV, respectively. Extraction recovery averaged 63.7% for I and 64.9% for II. This general assay methodology may be applied to assay small acidic peptides and peptidomimetics from biological fluids by LC/MS/MS.     

Simultaneous determination of Z-SU5416 and its interconvertible geometric E-isomer in rat plasma by LC/MS/MS

SU5416 is a selective inhibitor of vascular endothelial growth factor (VEGF) receptor, which plays a major role in vascular angiogenesis. SU5416 exists as the thermodynamically stable and pharmacologically active cis isomer (Z-isomer) in the solid state. In light-exposed solutions the unstable trans isomer (E-isomer) is formed. The E-isomer is unstable for synthesis and isolation and the analytical standard of the E-isomer is unavailable. A new, simple, fast and reliable LC/MS/MS method was developed to quantify both isomers simultaneously in rat plasma samples in order to support the study of disposition kinetics of Z- and E-SU5416. This method is sensitive (LOQ = 0.5 ng/ml), reproducible, and has a wide linear range (0.5-2500 ng/ml).     

Development and validation for high selective quantitative determination of metformin in human plasma by cation exchanging with normal-phase LC/MS/MS

An assay based on cation exchange solid-phase extraction and liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed for the quantitative determination of metformin in human plasma. The analytical method consists of cation exchange solid-phase extraction (VersaPlate CBA) without any further evaporation/dissolution steps and cation exchange-based HPLC separation (Capcell Pak SCX column) with a normal-phase gradient system followed by semi-micro LC/MS/MS in positive ion selected reaction monitoring mode using electrospray ionization. The method exhibited excellent performance in terms of selectivity, robustness, short run time (7 min/sample) and simplicity of sample preparation.

The calibration range was 10–1000 ng/ml with 0.2 ml of plasma. Intra- and inter-day mean accuracies were within the ranges of 100.3–105.0% and 101.2–105.3%, respectively. Intra- and inter-day precisions were within the ranges of 0.8–1.9% and 1.5–8.6%, respectively. Mean absolute recovery was 67.0% for metformin. No apparent loss of metformin after extraction was observed in an autosampler at 10 °C for 24 h. Dilution of metformin by blank human plasma up to 20-fold was tested and revealed no impact on the results of determination. Furthermore, the method exhibited high selectivity, since no effect on metformin analysis was observed on comparison of samples with or without nateglinide and other agents in plasma. Results obtained with the method were also comparable to a published LC–UV method on cross-validation.

This method can be applied to various clinical pharmacokinetic studies of metformin.     

LC／MS／MS法测定人血中硫普罗宁的浓度及药物动力学研究

目的建立LC／MS／MS法测定人血中硫普罗宁浓度的方法,并研究其在健康男性受试者体内的药物动力学。方法采用LC／MS／MS（ESI源）测定硫普罗宁的血浆浓度,计算药物动力学参数。结果硫普罗宁线性范围为25．0-5000ng·mL^-1,定量下限为25．0ng·mL^-1日内、日间精密度（RSD）均〈15％,准确度（RE）在15％以内。应用本法测得20名健康男性受试者口服200mg硫普罗宁胶囊后主要药代动力学参数为：tmax,为（4．20±1．01）h,t1／2为（5．61±4．42）h,Cmax为（4456±2447）ng·mL^-1,AU C0-24h为（20566±9902）ng·mL^-1,Ke为（0．173±0．094）h^-1。结论该法操作简便、快速、灵敏,可用于测定血浆中硫普罗宁浓度。     

The determination of RWJ-38705 (tramadol N-oxide) and its metabolites in preclinical pharmacokinetic studies using LC-MS/MS

A rapid and reliable analytical method is described for the simultaneous determination of RWJ-38705 (tramadol N-oxide) and several of its major metabolites in the plasma of Sprague-Dawley rats and Beagle dogs. Sample preparation using solid phase extraction was followed by reversed phase liquid chromatography (LC) coupled with tandem mass spectrometric (MS/MS) detection in the positive ionization mode. The assay was linear for all analytes over concentrations ranging from approximately 6 to 2000 ng/ml. The inter-assay reproducibility was generally less than 15% while accuracy values were within 13% of theoretical. The overall recovery of the analytes ranged from approximately 40 to 64% in rat plasma and 53-75% in dog plasma. This assay has proven to be sensitive, specific and reproducible, and it has been readily implemented in preclinical PK studies. Representative plasma concentration versus time profiles resulting from administration of TNO to rats and dogs are presented in this communication.     

Comparison of an ELISA and an LC/MS/MS method for measuring tacrolimus concentrations and making dosage decisions in transplant recipients

This study compared an enzyme-linked immunosorbent assay (ELISA) to a liquid chromatography-tandem mass spectrometry (LC/MS/MS) technique for measurement of tacrolimus concentrations in adult kidney and liver transplant recipients, and investigated how assay choice influenced pharmacokinetic parameter estimates and drug dosage decisions. Tacrolimus concentrations measured by both ELISA and LC/MS/MS from 29 kidney (n = 98 samples) and 27 liver (n = 97 samples) transplant recipients were used to evaluate the performance of these methods in the clinical setting. Tacrolimus concentrations measured by the two techniques were compared via regression analysis. Population pharmacokinetic models were developed independently using ELISA and LC/MS/MS data from 76 kidney recipients. Derived kinetic parameters were used to formulate "typical dosing" regimens for concentration targeting. Dosage recommendations for the two assays were compared. The relation between LC/MS/MS and ELISA measurements was best described by the regression equation ELISA = 1.02. (LC/MS/MS) + 0.14 in kidney recipients, and ELISA = 1.12. (LC/MS/MS) - 0.87 in liver recipients. ELISA displayed less accuracy than LC/MS/MS at lower tacrolimus concentrations. Population pharmacokinetic models based on ELISA and LC/MS/MS data were similar with residual random errors of 4.1 ng/mL and 3.7 ng/mL, respectively. Assay choice gave rise to dosage prediction differences ranging from 0% to 30%. ELISA measurements of tacrolimus are not automatically interchangeable with LC/MS/MS values. Assay differences were greatest in adult liver recipients, probably reflecting periods of liver dysfunction and impaired biliary secretion of metabolites. While the majority of data collected in this study suggested assay differences in adult kidney recipients were minimal, findings of ELISA dosage underpredictions of up to 25% in the long term must be investigated further.     

Simultaneous determination of Z-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (SU5416) and its interconvertible geometric isomer (SU5886) in rat plasma by LC/MS/MS

Z-3-[(2,4-Dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (SU5416) is a cytostatic substance in development as an anti-angiogenic agent. SU5416 exists as the thermodynamically stable cis or Z-isomer as a solid. Studies have shown that in light exposed solutions of SU5416, the unstable trans or E-isomer, namely SU5886, is formed. The E-isomer converts back to the Z-isomer when protected from light. The E-isomer is unstable for synthesis and isolation; therefore, the analytical standard of the E-isomer is not available. In this study, a simple, fast and reliable LC/MS/MS method has been developed to determinate both isomers simultaneously in rat plasma samples to support the study of disposition kinetics of SU5416. This method is sensitive (limit of quantitation (LOQ=0.5 ng/mL)), reproducible and has a wide linear range (0.5-2500 ng/mL). There was no conversion between E- and Z-isomer during sample preparation procedure and sample determination with LC/MS/MS. Experimental results proved that SU5416 and SU5886 have identical detection response. Therefore, SU5416 (Z-isomer) was used successfully as analytical standard for SU5886 (E-isomer). This method has been applied to rat plasma samples obtained from a pharmacokinetic study. This study underscores the use of LC/MS/MS technique for bioanalytical methods where analytical standards are not available and analytes are interconvertible.     

Determination of a beta(3)-agonist in human plasma by LC/MS/MS with semi-automated 48-well diatomaceous earth plate

Methods for the determination of a beta(3)-agonist (A) in human plasma were developed and compared based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection using a turbo ion spray (TIS) interface. Drug and internal standard were isolated from plasma by three sample preparation methods, liquid-liquid extraction, Chem Elut cartridges and 48-well diatomaceous earth plates, that successively improved sample throughput for LC/MS/MS. MS/MS detection was performed on a PE Sciex API 365 tandem mass spectrometer operated in positive ion mode and using multiple reaction monitoring (MRM). The precursor/product ion combinations of m/z 625/607 and 653/515 were used to quantify A and internal standard, respectively, after chromatographic separation of the analytes. Using liquid-liquid extraction and Chem Elut cartridges, the assay concentration range was 0.5-100 ng/ml. Using diatomaceous earth plates, the concentration range of the assay was extended to 0.5-200 ng/ml. For all three assays, the statistics for precision and accuracy is comparable. The assay accuracy ranged from 91-107% and intraday precision as measured by the coefficient of variation (CV) ranged 2-10%. The sample throughput was tripled when the diatomaceous earth plate method was compared with the original liquid-liquid extraction method.     

Quantitation of N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide and 4-amino-3,5-dichloropyridine in rat and mouse plasma by LC/MS/MS

The metabolism of N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (RP73401), a phosphodiesterase IV (PDE IV) inhibitor is extensive (unpublished); however, until recently, studies for this compound did not report 4-amino-3,5-dichloropyridine (ADCP) as a metabolite either in vitro or in vivo. This prompted a reinvestigation into the metabolism of RP73401 in rats and mice using mass spectrometry. The results of the reinvestigation confirmed that 4-amino-3,5-dichloropyridine was formed via the metabolism of RP73401 both in vitro and in vivo. In order to further investigate RP73401 hydrolysis in vivo, a liquid chromatography/mass spectrometry assay was developed and validated for the simultaneous determination of RP73401 and ADCP in rat and mouse plasma. The method used Waters Oasis HLB brand solid phase extraction cartridges to isolate the analytes (RP73401 and ADCP) and internal standard from the plasma. HPLC chromatographic separation was achieved using a Zorbax SB C18 HPLC column and detection was accomplished using positive ion atmospheric pressure chemical ionization tandem mass spectroscopy in multiple reaction monitoring (MRM) mode. The assay was developed and validated over the range of 0.5-100 ng ml(-1) for RP73401 and 5-500 ng ml(-1) for ADCP using 0.050 ml of plasma. The assay proved to be sensitive, accurate, precise and specific for RP73401 and ADCP. Intraday and interday quality control results routinely showed accuracy and precision to be within +/- 20%. This LC/MS/MS method was subsequently employed to investigate the hydrolysis of RP73401 in the rat and mouse, and determine the effects of tri-o-tolyl phosphate (TOTP, a carboxylesterase inhibitor) preadministration on the hydrolysis reaction in the rat.