Ternary-column system for high-throughput direct-injection bioanalysis by liquid chromatography/tandem mass spectrometry

As a continuation of our efforts to improve our high-flow on-line bioanalytical approach for high-throughput quantitation of drugs and metabolites in biological matrices by high-performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we have developed a ternary-column on-line LC/MS/MS system with dual extraction columns used in parallel for purification and an analytical column for analysis. The advantage of the dual extraction column system is that sample analysis can take place in one of the extraction columns while the other column is being equilibrated. Thus, the equilibration time does not add to the run time, hence shortening the injection cycle time and increasing the sample throughput. Moreover, the use of two extraction columns in parallel increases the number of samples that can be injected before the system fails due to an overused extraction column. Such a system has successfully been used to develop and validate a positive ion electrospray LC/MS/MS bioanalytical method for the quantitative determination of a guanidine-containing drug candidate in rat plasma. The system used for this work utilized two Oasis HLB extraction columns (1 x 50 mm, 30 microm), one C18 analytical column (3.9 x 50 mm, 5 microm), a ten-port switching value and a tandem mass spectrometer. The on-line analysis was accomplished by the direct injection of 10 microL of the sample, obtained by mixing a rat plasma sample 1:1 with an aqueous internal standard solution. Selected reaction monitoring (SRM) was utilized for the detection of the analyte and internal standard. The standard curve range was 1.00-200 ng/mL. The intra- and inter-day precision and accuracy were within 6.6%. The on-line purification step lasted for only 0.3 min and total run time was only 1.6 min.     

Simple and sensitive liquid chromatographic quantitative analysis of the novel marine anticancer drug Yondelis (ET-743, trabectedin) in human plasma using column switching and tandem mass spectrometric detection

The development of a simple and sensitive assay for the quantitative analysis of the marine anticancer agent Yondelis (ET-743, trabectedin) in human plasma using liquid chromatography (LC) with column switching and tandem mass spectrometric (MS/MS) detection is described. After protein precipitation with methanol, diluted extracts were injected on to a small LC column (10 x 3.0 mm i.d.) for on-line concentration and further clean-up of the sample. Next, the analyte and deuterated internal standard were back-flushed on to an analytical column for separation and subsequent detection in an API 2000 triple-quadrupole mass spectrometer. The lower limit of quantitation was 0.05 ng mL(-1) using 100 micro l of plasma with a linear dynamic range up to 2.5 ng ml(-1). Validation of the method was performed according to the most recent FDA guidelines for bioanalytical method validation. The time needed for off-line sample preparation has been reduced 10-fold compared with an existing LC/MS/MS method for ET-743 in human plasma, employing a labor-intensive solid-phase extraction procedure for sample pretreatment. The proposed column switching method was successfully applied in phase II clinical trials with Yondelis and pharmacokinetic monitoring.     

Quantitative determination of piritramide in human plasma and urine by off- and on-line solid-phase extraction liquid chromatography coupled to tandem mass spectrometry

A method for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) quantification of piritramide, a synthetic opioid, in plasma after conventional off-line solid-phase extraction (SPE) and in urine by on-line SPE-LC/MS/MS in positive electrospray mode was developed and validated. Applicability of the on-line approach for plasma samples was also tested. Deuterated piritramide served as internal standard. For the off-line SPE plasma method mixed cation-exchange SPE cartridges and a 150 x 2 mm C18 column with isocratic elution were used. For the on-line SPE method, a Waters Oasis HLB extraction column and the same C18 analytical column in a column-switching set-up with gradient elution were utilized. All assays were linear within a range of 0.5-100 ng/mL with a limit of detection of 0.05 ng/mL. The intra- and interday coefficients of variance ranged from 1.3 to 6.1% for plasma and 0.5 to 6.4% for urine, respectively. The extraction recovery for the off-line plasma assay was between 90.7 and 100.0%. Influence of matrix effects, and freeze/thaw and long-term stability were validated for both approaches; influence of urine pH additionally for quantification in urine.     

Determination of 2-methoxyestradiol in human plasma, using liquid chromatography/tandem mass spectrometry

A liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay was developed for the quantitative determination of 2-methoxyestradiol (2ME2) in human plasma. Sample pretreatment involved liquid-liquid extraction with ethyl acetate of 0.3-mL aliquots of plasma spiked with the internal standard, deuterated 2ME2 (2ME2-d5). Separation was achieved on a Zorbax Eclipse C18 column (2.1 x 50 mm, i.d., 5 microm) at room temperature using a gradient elution with methanol and water at a flow rate of 0.25 mL/min. Detection was performed using atmospheric pressure chemical ionization MS/MS by monitoring the ion transitions from m/z 303.1 --> 136.8 (2ME2) and m/z 308.1 --> 138.8 (2ME2-d5). Calibration curves were linear in the concentration range of 1-100 ng/mL. The accuracy and precision values, obtained from three different sets of quality control samples analyzed in quintuplicate on four separate occasions, ranged from 105-108% and from 3.62-5.68%, respectively. This assay was subsequently used for the determination of 2ME2 concentration in plasma of a patient with cancer after a single oral administration of 2ME2 at a dose of 2200 mg.     

Development of a rapid and sensitive liquid chromatography/tandem mass spectrometry method for the determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE), a novel anti-cancer agent in rat plasma

A rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method has been developed to determine 1, 2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE), a novel antineoplastic agent, in rat plasma. The analytes were separated on a C18 column with a mobile phase of methanol-water (75:25, v/v) and detected using a triple-quadrupole mass spectrometer in positive mode with the selective reaction monitoring. The characteristic ion dissociation transitions were m/z 603.0 --> 448.9 for derivatized BBSKE and m/z 631.0 --> 476.8 for derivatized internal standard. The assay was linear over a range of 1-1000 ng/mL with a lower limit of quantification of 1 ng/mL. Intra- and inter-day precisions were less than 9.6 and 5.0%, respectively, and the accuracy ranged from -5.2 to 4.0%. The validated method was successfully applied to the characterization of pharmacokinetic profile of BBSKE after oral administration in rats. Cop     

A novel on-line solid-phase extraction approach integrated with a monolithic column and tandem mass spectrometry for direct plasma analysis of multiple drugs and metabolites

An on-line solid-phase extraction liquid chromatography/tandem mass spectrometry (SPE LC/MS/MS) assay using a newly developed SPE column and a monolithic column was developed and validated for direct analysis of plasma samples containing multiple analytes. This assay was developed in an effort to increase bioanalysis throughput and reduce the complexity of on-line SPE LC/MS/MS systems. A simple column-switching configuration that requires only one six-port valve and one HPLC pumping system was employed for on-line plasma sample preparation and subsequent gradient chromatographic separation. The resulting analytical method couples the desired sensitivity with ease of use. The method was found to perform satisfactorily for direct plasma analysis with respect to assay linearity, specificity, sensitivity, precision, accuracy, carryover, and short-term stability of an eight-analyte mixture in plasma. A gradient LC condition was applied to separate the eight analytes that cannot be distinctly differentiated by MS/MS. With a run time for every injection of 2.8 min, a minimum of 300 direct plasma injections were made on one on-line SPE column without noticeable changes in system performance. Due to the ruggedness and simplicity of this system, generic methods can be easily developed and applied to analyze a wide variety of compounds in a high-throughput manner without laborious off-line sample preparation.     

Determination of acetylsalicylic acid and its major metabolite, salicylic acid, in human plasma using liquid chromatography-tandem mass spectrometry: application to pharmacokinetic study of Astrix in Korean healthy volunteers

The first liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for determination of acetylsalicylic acid (aspirin, ASA) and one of its major metabolites, salicylic acid (SA), in human plasma using simvastatin as an internal standard has been developed and validated. For ASA analysis, a plasma sample containing potassium fluoride was extracted using a mixture of ethyl acetate and diethyl ether in the presence of 0.5% formic acid. SA, a major metabolite of ASA, was extracted from plasma using protein precipitation with acetonitrile. The compounds were separated on a reversed-phase column with an isocratic mobile phase consisting of acetonitrile and water containing 0.1% formic acid (8:2, v/v). The ion transitions recorded in multiple reaction monitoring mode were m/z 179 --> 137, 137 --> 93 and 435 --> 319 for ASA, SA and IS, respectively. The coefficient of variation of the assay precision was less than 9.3%, and the accuracy exceeded 86.5%. The lower limits of quantification for ASA and SA were 5 and 50 ng/mL, respectively. The developed assay method was successfully applied for the evaluation of pharmacokinetics of ASA and SA after single oral administration of Astrix (entero-coated pellet, 100 mg of aspirin) to 10 Korean healthy male volunteers.     

Parallel extraction columns and parallel analytical columns coupled with liquid chromatography/tandem mass spectrometry for on-line simultaneous quantification of a drug candidate and its six metabolites in dog plasma.

A method with parallel extraction columns and parallel analytical columns (PEC-PAC) for on-line high-flow liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for simultaneous quantification of a drug candidate and its six metabolites in dog plasma. Two on-line extraction columns were used in parallel for sample extraction and two analytical columns were used in parallel for separation and analysis. The plasma samples, after addition of an internal standard solution, were directly injected onto the PEC-PAC system for purification and analysis. This method allowed the use of one of the extraction columns for analyte purification while the other was being equilibrated. Similarly, one of the analytical columns was employed to separate the analytes while the other was undergoing equilibration. Therefore, the time needed for re-conditioning both extraction and analytical columns was not added to the total analysis time, which resulted in a shorter run time and higher throughput. Moreover, the on-line column extraction LC/MS/MS method made it possible to extract and analyze all seven analytes simultaneously with good precision and accuracy despite their chemical class diversity that included primary, secondary and tertiary amines, an alcohol, an aldehyde and a carboxylic acid. The method was validated with the standard curve ranging from 5.00 to 5000 ng/mL. The intra- and inter-day precision was no more than 8% CV and the assay accuracy was between 95 and 107%.     

Liquid chromatography-tandem mass spectrometry for quantification of lacosamide, an antiepileptic drug, in rat plasma and its application to pharmacokinetic study

A rapid, simple and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) was developed for the determination of an antiepileptic drug, lacosamide, in rat plasma. The method involves the addition of acetonitrile and internal standard solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on column packed with octadecylsilica (5 µm, 2.0 × 50 mm) with 0.1% formic acid and acetonitrile as mobile phase, and the detection was performed on tandem mass spectrometry by the multiple‐reaction monitoring via an electrospray ionization source. The standard curve was linear over the concentration range from 0.3 to 1000 ng/mL. The lower limit of quantification was 0.3 ng/mL using 50 μL of rat plasma sample. The intra‐ and inter‐assay precision and accuracy were found to be less than 11.7 and 8.8%, respectively. The developed analytical method was successfully applied to the pharmacokinetic study of lacosamide in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of famotidine in low-volume human plasma by normal-phase liquid chromatography/tandem mass spectrometry

A rapid, sensitive and robust assay procedure using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the determination of famotidine in human plasma and urine is described. Famotidine and the internal standard were isolated from plasma samples by cation-exchange solid-phase extraction with benzenesulfonic acid (SCX) cartridges. The urine assay used direct injection of a diluted urine sample. The chromatographic separation was accomplished by using a BDS Hypersil silica column with a mobile phase of acetonitrile-water containing trifluoroacetic acid. The MS/MS detection of the analytes was set in the positive ionization mode using electrospray ionization for sample introduction. The analyte and internal standard precursor-product ion combinations were monitored in the multiple-reaction monitoring mode. Assay calibration curves were linear in the concentration range 0.5--500 ng ml(-1) and 0.05--50 microg ml(-1) in plasma and urine, respectively. For the plasma assay, a 100 microl sample aliquot was subjected to extraction. To perform the urine assay, a 50 microl sample aliquot was used. The intra-day relative standard deviations at all concentration levels were <10%. The inter-day consistency was assessed by running quality control samples during each daily run. The limit of quantification was 0.5 ng ml(-1) in plasma and 0.05 microg ml(-1) in urine. The methods were utilized to support clinical pharmacokinetic studies in infants aged 0-12 months.     

Quantification of lurasidone, an atypical antipsychotic drug, in rat plasma with high-performance liquid chromatography with tandem mass spectrometry

A liquid chromatography–tandem mass spectrometric (LC/MS/MS) method was developed for the determination of an atypical antipsychotic drug, lurasidone, in rat plasma. The method involves the addition of acetonitrile and ziprasidone (internal standard) solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on a column packed with octadecylsilica (5 μm, 2.0 × 50 mm) with 0.1% formic acid and 0.1% formic acid in acetonitrile as mobile phase and the detection was performed using tandem mass spectrometry by multiple‐reaction monitoring via an electrospray ionization source. The standard curve was linear (r = 0.9982) over the concentration range 0.002–1 μg/mL. The intra‐ and inter‐assay precisions were 1.7 and 8.6%, respectively. The accuracy range was from 90.3 to 101.8%. The lower limit of quantification was 2.0 ng/mL using 50 μL of rat plasma sample. The developed analytical method was successfully applied to the pharmacokinetic study of lurasidone in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Method development for the concentration determination of a protein in human plasma utilizing 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometric detection

Although liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology has been widely used for quantitative analysis of small organic molecules, it has been a challenging task to quantitatively analyze protein samples utilizing this technology in biological matrices for pre-clinical and clinical studies. Here we present our initial results in method development for the quantitative determination of rK5 protein concentrations in human plasma samples utilizing LC/MS/MS technology. A protein similar in structure to rK5, but with a slightly reduced molecular weight, was used as internal standard. A 96-well solid-phase extraction procedure was developed to effectively extract protein analytes from plasma samples. Quantitative analysis was obtained by a novel approach of protein monitoring that employed selective reaction monitoring (SRM). Even though mass spectrometry of the internal standard protein gave no fragment ions, SRM monitoring greatly reduced background interference. Using samples prepared in human plasma with sodium EDTA as anticoagulant, a correlation coefficient (r(2)) of 0.9940 was obtained by producing a single standard curve with the injection of six rows of standards with a concentration range from 100 ng/mL to 10 microg/mL. The mean analytical recovery for these standards ranged from 91.5 to 103.6%. The CVs for individual standard levels ranged from 3.7 to 20.9%. The experiment was also repeated using samples prepared in human plasma with sodium heparin as anticoagulant, which produced a correlation coefficient (r(2)) of 0.9952 obtained from a single standard curve with the injection of four rows of standards with a concentration range from 50 ng/mL to 10 microg/mL. The mean analytical recovery for the standards ranged from 96.2 to 104.6%. The CVs for individual standard levels ranged from 2.6 to 15.6%.     

Direct injection versus liquid-liquid extraction for plasma sample analysis by high performance liquid chromatography with tandem mass spectrometry.

Direct injection versus liquid-liquid extraction for post-dose human plasma sample analysis by high performance liquid chromatography with tandem mass spectrometry (LC/MS/MS) have been studied using a drug candidate compound. For the direct-injection method, an Oasis(R) HLB column (1 x 50 mm, 30 micrometer) was used as the on-line extraction column and a conventional Waters symmetry C18 column (3.9 x 50 mm, 5 micrometer) was used as the analytical column. Each plasma sample (100 microL) was mixed with 100 microL of a working solution of the internal standard in aqueous 0.05 M ammonium acetate (pH 6.9), and portions (10 microL) of these samples were then injected into the LC/MS/MS system. For the liquid-liquid extraction method, a YMC Basic C18 column (2.0 x 50 mm, 5 micrometer) was used as the analytical column. Each sample (0.5 mL) was extracted with methyl tert-butyl ether and the extract was reconstituted and injected into the LC/MS/MS system. The total analysis time for both methods was 2.0 min per sample. The accuracy, inter-day precision and intra-day precision obtained from the quality control samples were within 8% for both methods. The analysis results of post-dose human plasma samples showed that the deviations of 91% of the concentrations obtained using the direct-injection method were within +/-20% from the concentrations obtained using the liquid-liquid extraction method, and the overall average percentage deviation was -1.5%. The results showed that the two methods were equivalent in terms of total chromatographic run time, accuracy and precision. However, for a batch of 100 samples, the sample preparation time for the direct-injection method was only about 25% of the time required for liquid-liquid extraction. This decrease in sample preparation time resulted in the doubling of the overall sample analysis throughput.     

Simultaneous determination of docetaxel and ketoconazole in rat plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for simultaneous quantification of docetaxel and ketoconazole in rat plasma with paclitaxel as internal standard (IS). The analytes were extracted from rat plasma by using a liquid-liquid extraction technique with ethyl acetate and the LC separation was performed on a Cosmosil-C(18) analytical column (150 mm x 2.0 mm i.d., Nacalai Tesque Inc., Japan). The extracted samples were analyzed with LC/MS/MS, operating in selected reaction monitoring (SRM) mode. The SRM transitions of precursor ions to product ions were 830.3-->549.1 (m/z) for docetaxel, 531.2-->489.3 (m/z) for ketoconazole, and 876.7-->307.9 (m/z) for the IS. The calibration curves were linear over the range of 2-500 ng/mL for docetaxel and 50-20 000 ng/mL for ketoconazole, with coefficients of correlation above 0.999. The limits of quantification for docetaxel and ketoconazole were both 2 ng/mL. The limit of detection for each analyte was 1 ng/mL. The intra- and inter-day precision (CV) of analysis were within 7%, and the accuracy ranged from 95 to 110%. The overall recoveries for docetaxel and ketoconazole were about 89.0% and 91.1%, respectively. The total analysis time was only 9.0 min. This quantitation method was successfully applied to the simultaneous determination of docetaxel and ketoconazole in rat plasma and some potential interaction was found in the current coadministration pharmacokinetic study. This established method was also utilized in the in vitro and in vivo drug-drug interaction study of docetaxel and ketoconazole (to be published).     

Determination of telmisartan in human blood plasma: Part II: Liquid chromatography-tandem mass spectrometry method development, comparison to immunoassay and pharmacokinetic study

A new liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) method with on-line sample clean-up for the determination of telmisartan in human blood plasma is presented. This technique is compared to a previously introduced enzyme-linked immunosorbent assay (ELISA), where fluorescence is used as detection method. For the LC/MS method applying an internal calibration via a deuterated internal standard, the limit of detection was 0.3 ng/mL, the limit of quantification was 0.9 ng/mL and the linear range extended from 0.9 to 1000 ng/mL. Forty-eight plasma samples from four healthy volunteers were analyzed in a pharmacokinetic study to obtain data for the method comparison. As a result, these two new and independent analytical methods for the determination of telmisartan in human blood plasma proved to yield comparable results for the amount of analyte.     

Enantiomeric separation and quantification of fluoxetine (Prozac) in human plasma by liquid chromatography/tandem mass spectrometry using liquid-liquid extraction in 96-well plate format

Fluoxetine (Prozac) is currently one of the widely prescribed selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression. A high-throughput sample preparation procedure using liquid-liquid extraction (LLE) in a 96-well plate format in conjunction with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for quantification of fluoxetine enantiomers in human plasma. After addition of internal standard and ammonium hydroxide, samples were extracted with ethyl acetate. The organic extract was evaporated to dryness and reconstituted in methanol. Where possible, sample transfer and LLE steps were automated using a Tomtec Quadra 96 workstation. Adequate separation of fluoxetine enantiomeric pairs (resolution of 1.17) was achieved on a vancomycin column eluted with methanol containing 0.075% (by weight) ammonium trifluoroacetate. A triple quadrupole mass spectrometer, operated in the multiple reaction monitoring mode at m/z 310-->44 for fluoxetine enantiomeric pairs and m/z 287-->241 for oxazepam (internal standard), was used. Analysis was performed in the positive ion mode using atmospheric pressure chemical ionization (APCI). The standard curve range was 2.0-1000 ng/mL for each fluoxetine enantiomer. The intra- and inter-day precision and accuracy of the quality control (QC) samples were <12.5% (CV) and <13.6% (CV), respectively, for each fluoxetine enantiomer; the correlation coefficient was >0.990. Method ruggedness was demonstrated by the reproducible performance of the assay during a three-day validation period.     

液相色谱同位素稀释串联质谱法测定人血清中17β-雌二醇含量

研究建立了以人血清中E2-16,16,17-d3为内标测定17β-雌二醇的液相色谱/串联质谱(ID-LC/MS/MS)方法。血清样品经固相萃取装置(SPE)提取雌二醇,乙酸乙酯萃取净化,吹干复溶后用10-乙基吖啶酮-2-磺酰氯(EASC)进行衍生。以Agilent Eclipse XDB-C18色谱分离柱,乙腈、水梯度洗脱,使用电喷雾三重四极杆串联质谱的多重反应监测模式测定,以校准曲线法进行定量。所建立的液相色谱同位素稀释串联质谱法(ID-LC/MS/MS)对于分析血清17β-雌二醇的批内、批间RSD分别为0.29%～0.73%和0.18%～0.28%,回收率为99.6%～100.2%,采用IFCC RELA比对(JCTLM比对)样品进行了方法比较,测定结果与其他实验室相比偏差在0.8%范围内。方法可作为人血清中17β-雌二醇含量测量参考方法。     

Determination of roxatidine in human plasma by liquid chromatography/electrospray mass spectrometry and application to a clinical pharmacokinetic study

A rapid and sensitive liquid chromatography/mass spectrometry (LC/MS) method was developed and validated for the determination of roxatidine in human plasma. Roxatidine was extracted by single liquid-liquid extraction with tert-butyl methyl ether, and the chromatographic separation was performed on a C8 column. The total analytical run time was relatively short (5 min), and the limit of assay quantification was 2 ng/mL using 0.1 mL of human plasma. Roxatidine and the internal standard, propranolol, were monitored in selected ion monitoring (SIM) mode at m/z 307.3 and 260.3, respectively. The standard curve was linear over a concentration range from 2-500 ng/mL, and the correlation coefficients were >0.999. The mean intra- and inter-day assay accuracy ranged from 103.4-108.8% and 102.3-110.0%, respectively, and the mean intra- and inter-day precision was between 3.3-8.8% and 5.3-6.2%, respectively. The developed assay method was successfully applied to a pharmacokinetic study in human volunteers after oral administration of roxatidine acetate hydrochloride at a dose of 75 mg.     

Validated assay for the simultaneous quantification of total vincristine and actinomycin‐D concentrations in human EDTA plasma and of vincristine concentrations in human plasma ultrafiltrate by high‐performance liquid chromatography coupled with tandem mass spectrometry

A sensitive, specific and efficient high‐performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the simultaneous determination of total vincristine and actinomycin‐D concentrations in human plasma and an assay for the determination of unbound vincristine are presented. Electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and heated electrospray ionization (H‐ESI) were tested as ionization interfaces. For reasons of robustness ESI was chosen followed by tandem mass spectrometry (ESI‐MS/MS). For the plasma assay a 30 µL aliquot was protein precipitated with acetonitrile/methanol (50:50, v/v) containing the internal standard vinorelbine and 10 µL volumes were injected onto the HPLC system. To determine unbound vincristine, ultrafiltrate was produced from plasma using 30 kDa centrifugal filter units. The plasma ultrafiltrate was mixed with methanol (50:50, v/v), internal standard vinorelbine was added and 20 µL aliquots were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm i.d. Xbridge C₁₈ column using 1 mM ammonium acetate/acetonitrile (30:70, v/v) adjusted to pH 10.5 with ammonia, run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies in plasma vincristine from 0.25 to 100 ng/mL and actinomycin‐D from 0.5 to 250 ng/mL using plasma sample volumes of only 30 µL. Vincristine in plasma ultrafiltrate can be quantified from 1 to 100 ng/mL. Validation results demonstrate that vincristine and actinomycin‐D can be accurately and precisely quantified in human plasma and plasma ultrafiltrate with the presented methods. The assays are now in use to support clinical pharmacological studies in children treated with vincristine and actinomycin‐D. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative analysis of cimetidine in human plasma using LC/APCI/SRM/MS.

A quantitative method was developed and validated for rapid and sensitive analysis of cimetidine in human plasma. The method involved the use of liquid chromatography (LC) coupled with atmospheric pressure chemical ionization (APCI) and selected reaction monitoring (SRM) mass spectrometry (MS). A cimetidine analog, SKF92374, was used as the internal standard. Separation of cimetidine and the internal standard was accomplished using a reverse-phase HPLC column (C18). The eluted components were ionized by the APCI source and subsequently detected by a highly selective triple quadrupole mass spectrometer in the SRM mode. Linear standard curves were obtained from 5 ng/mL (lower limit of quantitation) to 10,000 ng/mL. The results demonstrated excellent precision (%RSD 1. 1-8.9%) and accuracy (94.7-108.0%) over this range. In addition, the amount of plasma sample needed for analysis was small (50 muL), and the plasma pretreatment (analyte recovery >94%) was simple and time saving. This assay was used to evaluate cimetidine levels in premature infants following intravenous infusion of cimetidine.