Validation of a sensitive LC/MS/MS method for simultaneous quantitation of flupentixol and melitracene in human plasma

A sensitive method has been developed and validated, using LC/ESI-MS/MS, for simultaneous quantitation of flupentixol and melitracen—antidepressant drugs, in human plasma. The quantitation of the target compounds was determined in a positive ion mode and multiple reaction monitoring (MRM). The method involved a repeated liquid–liquid extraction with diethyl ether and analytes were chromatographed on a C₈ chromatographic column by elution with acetonitrile–water–formic acid (36:64:1, v/v/v) and analyzed by tandem mass spectrometry. The method was validated over the concentration ranges of 26.1–2090 pg/ml for flupentixol and 0.206–4120 ng/ml for melitracen. The correlation coefficients of both analyst were >0.998 for six sets of calibration curves. The recovery was 60.9–75.1% for flupentixol, melitracen and internal standard. The lower limit of quantitation (LLOQ) detection was 26.1 pg/ml for flupentixol and 0.206 ng/ml for melitracen. Intra- and inter-day precision of the assay at three concentrations were 2.15–5.92% with accuracy of 97.6–103.0% for flupentixol and 0.5–6.36% with accuracy of 98.7–101.7% for melitracen. Stability of compounds was established in a battery of stability studies, i.e., bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for bioequivalence study of flupentixol and melitracen in healthy human male volunteers.     

Liquid-liquid extraction of strongly protein bound BMS-299897 from human plasma and cerebrospinal fluid, followed by high-performance liquid chromatography/tandem mass spectrometry

BMS-299897 is a γ-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Liquid–liquid extraction (LLE), chromatographic/tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for the quantitation of BMS-299897 in human plasma and cerebrospinal fluid (CSF). Both methods utilized ¹³C₆-BMS-299897, the stable label isotope analog, as the internal standard. For the human plasma extraction method, two incubation steps were required after the addition of 5 mM ammonium acetate and the internal standard in acetonitrile to release the analyte bound to proteins prior to LLE with toluene. For the human CSF extraction method, after the addition of 0.5 N HCl and the internal standard, CSF samples were extracted with toluene and no incubation was required. The organic layers obtained from both extraction methods were removed and evaporated to dryness. The residues were reconstituted and injected into the LC/MS/MS system. Chromatographic separation was achieved isocratically on a MetaChem C18 Hypersil BDS column (2.0 mm × 50 mm, 3 μm). The mobile phase contained 10 mM ammonium acetate pH 5 and acetonitrile. Detection was by negative ion electrospray tandem mass spectrometry. The standard curves ranged from 1 to 1000 ng/ml for human plasma and 0.25–100 ng/ml for human CSF. Both standard curves were fitted to a 1/x weighted quadratic regression model. For both methods, the intra-assay precision was within 8.2% CV, the inter-assay precision was within 5.4% CV, and assay accuracy was within ±7.4% of the nominal values. The validation and sample analysis results demonstrated that both methods had acceptable precision and accuracy across the calibration ranges.     

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.     

Simultaneous determination of niacin, niacinamide and nicotinuric acid in human plasma

A sensitive, specific, accurate, and reproducible HPLC/MS-method for the simultaneous quantitative determination of niacin (NA) and its main metabolites niacinamide (NAM) and nicotinuric acid (NUR) in human plasma using chinolin-3-carboxylic acid as an internal standard was developed and validated according to international guidelines for method validation.

All analytes and the internal standard were separated from acidified plasma by solid phase extraction. Afterwards the extracted samples were analyzed by HPLC/MS in the positive electrospray ionization mode (ESI) and selected ion monitoring (SIM). The total run time was 7 min between injections. The assay had a lower limit of quantification of 50.0 ng/mL for each analyte using 1 mL of plasma. The calibration curves were linear in the measured range between 50.0 and 750 ng/mL plasma. The overall precision and accuracy for all concentrations of quality controls and standards was better than 15%. No indications were found for possible instabilities of niacin, niacinamide and nicotinuric acid in plasma at −20 °C, in the extraction solvent or after repeated thawing/freezing cycles. In stabilities were observed in whole blood and in plasma at room temperature. The recovery of the extraction method ranged from 86 to 89% for the three analytes.     

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.     

High-throughput quantitation of nefazodone and its metabolites in human plasma by high flow direct-injection LC-MS/MS

A rapid, selective and sensitive high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method coupled with high flow direct-injection on-line extraction has been developed and validated for the simultaneous quantitation of nefazodone and its three active metabolites, hydroxynefazodone, triazole-dione (BMS-180492) and m-chlorophyenylpiperazine (mCPP) in human plasma. The method utilized d₇-nefazodone, d₇-hydroxynefazodone, d₄-BMS-180492 and d₄-mCPP as internal standards (IS). The plasma samples were injected into the LC–MS/MS system after simply adding the internal standard solution and centrifuging. The required extraction and chromatographic separation of the analytes were achieved on an Oasis^® HLB column (on-line extraction column, 1 mm × 50 mm, 30 μm) and a conventional Luna C8 column (analytical column, 4.6 mm × 50 mm, 5 μm). Detection was by positive ion electrospray tandem mass spectrometry. The total analysis run time for each sample was 2 min, which included the time needed for on-line extraction, chromatographic separation and LC–MS/MS analysis. The assay was validated for each analyte and the concentrations ranged from 2.0 to 500 ng/ml for nefazodone, hydroxynefazodone and mCPP and from 4.0 to 1000 ng/ml for BMS-180492, respectively. The assay was used for the high-throughput sample analysis of thousands of pharmacokinetic study samples and was proven to be rapid, accurate, precise, sensitive, specific and rugged.     

Quantitation of Armillarisin A in human plasma by liquid chromatography-electrospray tandem mass spectrometry

A rapid and sensitive LC–MS/MS method for quantifying Armillarisin A in human plasma after a single oral dose (40 mg) has been developed and validated. Sample preparation used liquid–liquid extraction with a mixture of diethyl ether–dichloromethane (60:40, v/v) in an acidic environment. The retention times of Armillarisin A and the internal standard, probenecid, were 1.63 and 1.78 min, respectively. The calibration curve was linear over the range 0.15–50 ng/mL with a limit of quantitation of 0.15 ng/mL. The coefficient of variation as a measure of intra- and inter-day precision was <9.3% and the accuracy was in the range 92.5–108.0%. The Armillarisin A concentration–time profile in human plasma was determined after an oral dose of a 40 mg tablet.     

A direct LC/MS/MS method for the determination of ciclopirox penetration across human nail plate in in vitro penetration studies

Due to severe chelating effect caused by N-hydroxylpyridone group of ciclopirox, there is no published direct HPLC or LC/MS/MS method for the determination of ciclopirox in any in vitro or in vivo matrix. Instead, the time-consuming pre-column derivatization methods have been adapted for indirect analysis of ciclopirox. After overcoming the chelating problem by using K₂EDTA coated tubes, a direct, sensitive and high-throughput LC/MS/MS method was successfully developed and validated to determine the amount of ciclopirox that penetrated across the nail plate during in vitro nail penetration studies. The method involved adding a chemical analog, chloridazon as internal standard (IS) in K₂EDTA coated tubes, mixing IS with ciclopirox in a 96-well plate and then proceeding to LC/MS/MS analysis. The MS/MS was selected to monitor m/z 208.0 → 135.8 and 221.8 → 77.0 for ciclopirox and IS, respectively, using positive electrospray ionization. The method was validated over a concentration range of 8–256 ng/mL, yielding calibration curves with correlation coefficients greater than 0.9991 with a lower limit of quantitation (LLOQ) of 8 ng/mL. The assay precision and accuracy were evaluated using quality control (QC) samples at three concentration levels. Analyzed concentrations ranged from 101% to 113% of their respective nominal concentration levels with coefficients of variation (CV) below 10.6%. The average recovery of ciclopirox from nail matrix was 101%.     

LC-MS/MS快速测定大鼠血浆中HHY-002浓度

目的：建立用于测定治疗慢性心律失常药物HHY-002的大鼠血浆测定法。方法：流动相为乙腈-0．1％甲酸（65：35）,流速0．200mL／min,色谱柱Phenomenex LunaC18柱（150mm×2．00mm,5μm,5micron）,进样量为10μL,柱温35℃,以地西泮为内标。结果：HHY-002在1．98～1013．50ng／mL的范围内呈良好的线性关系（r=0．998）,最低定量限达1．98ng／mL,绝对回收率高于80％,日内和日间误差小于10％,方法回收率大于（81．4±4．5）％,符合生物样品分析要求。结论：建立的LC—MS／MS方法专属性强,灵敏度高,可用于HHY-002的体内定量分析。     

A direct determination of thymidine triphosphate concentrations without dephosphorylation in peripheral blood mononuclear cells by LC/MS/MS

A rapid, sensitive and specific analytical method with minimal sample preparation for the measurement of thymidine triphosphate (TTP) in peripheral blood mononuclear cells (PBMC) by LC/MS/MS has been developed. PBMC were separated from whole blood or buffy coat. The analyte and internal standard were extracted from PBMC with 70% methanol (pH 7.2). These extracts after centrifugation were directly injected onto LC/MS/MS without need for any further sample preparation. The calibration curve was linear over the range 0.8–800 ng/ml. Mean inter- and intra-assay coefficients of variation (CVs) over the range of the standard curve were less than 10%. The overall recovery of TTP was 103.5%.     

LC/ESI-MS method for the determination of trimetazidine in human plasma: application to a bioequivalence study on Chinese volunteers

A rapid liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) method with good sensitivity and specificity has been developed and validated for the identification and quantification of trimetazidine in human plasma. Trimetazidine and lidocaine (internal standard) were isolated from plasma samples by protein precipitation with methanol. The chromatographic separation was accomplished on a Xterra MS C₁₈ Column (150 mm × 4.6 mm, 5 μm particle size) with the mobile phase consisting of methanol and water (40:60, v/v) (pH 2.0, adjusted with trifluoroacetic acid), and the flow rate was set at 0.6 mL/min. Detection was performed on a single quadruple mass spectrometer by selected ion monitoring (SIM) mode (m/z 267.0 for trimetazidine and m/z 235.0 for lidocaine) with the retention time at about 3.47 and 5.05 min, respectively. The calibration curve for trimetazidine was satisfactory with regression coefficient 0.9995 over the range of 2.5–100 ng/mL in the plasma. The LOQ (S/N = 10) was accordingly 2.5 ng/mL. The intra-day and inter-day precision expressed as relative standard deviation was 2.83–6.10% and 4.83–5.82%. The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test versus reference product) in 19 healthy male Chinese volunteers. After a single 20 mg dose for the test and reference product, the resulting mean of major pharmacokinetic parameters such as AUC_0–24, AUC_0−∞, C_max, T_max and t_1/2 of trimetazidine were (673.1 ± 117.6 ng h mL⁻¹ versus 652.3 ± 121.9 ng h mL⁻¹), (717.1 ± 120.9 ng h mL⁻¹ versus 692 ± 128.6 ng h mL⁻¹), (74.85 ± 12.13 ng mL⁻¹ versus 71.93 ± 14.32 ng mL⁻¹), (2.312 ± 0.663 h versus 2.211 ± 0.608 h) and (4.785 ± 0.919 h versus 4.740 ± 0.823 h), respectively, indicating that these two kinds of tablets were bioequivalent in the Chinese population.     

LC/MS/MS quantitation of an anti-cancer drug in human plasma using a solid-phase extraction workstation: application to population pharmacokinetics

A liquid chromatographic/mass spectrometric (LC/MS/MS) method to quantitate an anti-cancer drug in human plasma was validated. The method has proven suitable for routine quantitation of the experimental anti-cancer compound at concentrations from 1 to 400 ng/ml. Retention times of the compound and internal standard (compounds I and II, respectively) were 1.8 and 2.1 min, respectively. No interfering endogenous peaks were observed throughout the validation process. Precision estimates for this approach were typically less than 5% relative standard deviation (RSD) across the calibration range. Other validation parameters studied included specificity, system reproducibility, limit of quantitation, accuracy, linear range, and stability of the compound and internal standard in plasma and injection solvent. This method was used to quantify drug for population pharmacokinetic studies.     

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.     

Validated HPLC-MS/MS method for determination of quetiapine in human plasma

A validated, highly sensitive and selective high-pressure liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitative determination of quetiapine (QUE) in human Na2EDTA plasma with mass spectrometry (MS) detection. Clozapine (CLO) was employed as an internal standard. Samples were extracted using solid phase extraction (SPE). Oasis HLB cartridges and the concentration of quetiapine was determined by isocratic HPLC-MS/MS. The SRM mode was used for MS/MS detection. The method was validated over a concentration range of 1.0-382.2 ng/mL. Inter- and intra-day precision and accuracy of the proposed method were characterized by relative standard deviation (R.S.D.) and the percentage of deviation, respectively; both were lower than 8%. The developed method was employed in the pharmacokinetic study of quetiapine.     

A selective and rapid method for the quantification of captopril in human plasma using liquid chromatography/selected reaction monitoring mass spectrometry

A specific hyphenated high performance liquid chromatography–mass spectrometric (LC–MS/MS) assay was developed for the determination of captopril in plasma. The drug was extracted from plasma using liquid–liquid extraction with a mixture of diethylether:dichloromethane. After the addition of the internal standard, samples were applied to a prepacked C₈ Waters Symmetry column. The ion trap MS/MS detector was equipped with electrospray ionization (ESI) source operating in the positive ion mode. Drug determination was accomplished monitoring captopril at molecular ion m/z 218 and MS/MS (daughter) at m/z 171.6. The method was applied to captopril determination in human plasma after the administration of captopril 50 mg tablets to healthy volunteers who have participated in a pharmacokinetic study.

The method was proved to be specific and precise by testing six different plasma batches. Linearity was established for the range of concentrations 25–3000 ng/ml with a regression factor of 0.9995. Intra-day accuracy ranged from 90.16 to 96.18%, while the intra-day precision ranged from 2.60 to 9.66% at the concentrations of 75, 1440 and 2500 ng/ml. Inter-day precision of the method ranged from 5.04 to 10.10%. This validated method of analysis was successfully applied to human plasma analyses after the administration of a single dose of 50 mg captopril tablets to healthy volunteers.     

Development and validation of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of sunitinib in rabbit plasma

A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the measurement of sunitinib in rabbit plasma. After protein precipitation with acetonitrile, samples were analyzed on a Zorbax Extend-C₁₈ column (150 mm×4.6 mm, 5μm). The mobile phase consisted of a mixture of acetonitrile and deionized water (containing 0.05% formic acid)at a ratio of 27:73 (v/v), and the flow rate was set at 0.8 mL/min.The column temperature was maintained at 30 ^oC. The LC eluate was detected by an electrospray ionization (ESI) source operated in the positive ion mode, and quantification was conducted using MRM of the transitions m/z 399.24→283.01 and m/z 415.19→178.00 for sunitinib and internal standard (IS, diltiazem hydrochloride), respectively. The calibration curve was linear in the range of 2–600 ng/mL. The lower limit of quantification was 2 ng/mL. The method also exhibited satisfactory results in terms of sensitivity, specificity, accuracy (with relative error ranging from –4.0% to 1.1%), precision (with intra- and inter-day relative standard deviations ranging from 2.8% to 9.5%),matrix effect, recovery as well as stability. Taken together, our newly developed method was reliable to monitor sunitinib concentrations in rabbit plasma.     

Determination of limonin in rat plasma by liquid chromatography-electrospray mass spectrometry

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for determination of limonin (LM) in rat plasma has been developed and validated. The method had advantages of a single liquid–liquid extraction with ether and high sensitivity. Analyses were conducted at a flow rate of 0.2 ml/min by a gradient elution. The detection utilized selected ion monitoring in the negative ion mode at m/z 460.00 and 423.15 for the deprotonated molecular ions of LM and the internal standard, respectively. The quantitation limit for LM in rat plasma was 1.0 ng/ml. The linearity was also excellent over the concentration range of 1.9–500 ng/ml of LM. The intra- and inter-day precision (relative standard deviation (R.S.D.%)) was lower than 10% and accuracy ranged from 90 to 110%, showing a good reproducibility. This developed method was successfully applied to analysis of LM in biological fluids.     

A liquid chromatography tandem mass spectrometry method for the quantification of indocyanine green in dog plasma and bile

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of indocyanine green (ICG) in dog plasma and bile. An ICG analog (IR-820) was used as an internal standard. A protein precipitation method was used with acetonitrile for plasma sample preparation, whereas bile samples were diluted with water (120-fold) prior to analysis. Using MS/MS in the multiple reaction monitoring mode, ICG and IR-820 were detected in both matrices without interference. The lower limit of quantitation for ICG in dog plasma was 3 ng/mL, with an intra- and inter-day accuracy (%Bias) and precision (%CV) of less than 15%, so it was possible to study the pharmacokinetics of ICG in bile duct-cannulated dogs and assess their liver function after surgery. The method described herein is sensitive, selective and faster than other existing methods (e.g., spectrophotometry, HPLC/UV–vis detection, or HPLC/fluorescence detection).     

A sensitive human bone assay for quantitation of tigecycline using LC/MS/MS

Tigecycline (Tygacil,Wyeth) is a first-in-class, broad spectrum antibiotic with activity against multiple-resistant organisms. In order to address the unexpectedly low tigecycline concentrations in human bone samples analyzed using a LC/MS/MS method developed elsewhere, we have developed and validated a new and sensitive human bone assay for the quantitation of tigecycline using LC/MS/MS. The new method utilizes the addition of a stabilizing agent to the human bone sample, homogenization of human bone in a strong acidic-methanol extraction solvent, centrifugation of the bone suspension, separation by liquid chromatography, and detection of tigecycline by mass spectrometry. Linearity was demonstrated over the concentration range from 50 to 20,000 ng/g using a 0.1g human bone sample. The intra- and inter-day accuracy of the assay was within 100+/-15%, and the corresponding precision (CV) was <15%. The stability of tigecycline was evaluated and shown to be acceptable under the assay conditions. The extraction recovery of tigecycline with the current method was 79% when using radio-labeled rat bone samples as a substitute for human bone samples. Twenty-four human bone samples collected previously from volunteers without infections who had elective orthopedic surgery after receiving a single dose of tigecycline were re-analyzed using the current validated method. Tigecycline concentrations in these samples ranged from 238 to 794 ng/g with a mean value 9 times higher than the mean concentration previously reported. The data demonstrated that the current method has significantly higher extraction efficiency than the previously reported method.