Rapid multiresidue determination of organochlorine and organophosphorus compounds in human serum by solid-phase extraction and gas chromatography coupled to tandem mass spectrometry

A rapid analytical method for the multiresidue determination of several organochlorine and organophosphorus pesticides and polychlorinated biphenyls in human serum samples has been developed. Analytes were isolated by solid-phase extraction using C18 cartridges with subsequent analysis by GC-MS/MS using a glass liner packed with CarboFrit in the GC injection port. Labelled surrogate internal standards (fenitrothion D6, HCB (13)C6, p, p'-DDE D8 and PCB 138 (13)C12) were added to the samples before the extraction and were used for quantitation and for quality control in the analysis of real-world samples. Accuracy and precision were evaluated by using serum samples fortified at two concentration levels for the three families of compounds, with satisfactory results in the majority of cases. The high selectivity and sensitivity of GC-MS/MS allowed low detection limits of 0.05-0.5 ng mL(-1) for most of the analytes investigated. The developed procedure improves other current methodologies for the analysis of pesticides and PCBs in biological fluids, especially as regards to analysis time and simplicity of sample treatment. The method was applied to several serum samples obtained from farmers devoted to citrus crop production. Chlorpyrifos, HCB, p, p'-DDE and the higher chlorinated PCBs (153, 138 and 180) were the most frequently detected compounds.     

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.     

Determination of diazepam and its metabolites in human urine by liquid chromatography/tandem mass spectrometry using a hydrophilic polymer column

Diazepam and its major metabolites, nordazepam, temazepam and oxazepam, in human urine samples, were analyzed by liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using a hydrophilic polymer column (MSpak GF-310 4B), which enables direct injection of crude biological samples. Matrix compounds in urine were eluted first from the column, while the target compounds were retained on the polymer stationary phase. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All compounds showed base-peak ions due to [M+H]+ ions on LC/MS with positive ion electrospray ionization, and product ions were produced from each [M+H]+ ion by LC/MS/MS. Quantification was performed by selected reaction monitoring. All compounds spiked into urine showed method recoveries of 50.1-82.0%. The regression equations for all compounds showed excellent linearity in the range of 0.5-500 ng/mL of urine. The limits of detection and quantification for each compound were 0.1 and 0.5 ng/mL of urine, respectively. The intra- and inter-day coefficients of variation for all compounds in urine were not greater than 9.6%. The data obtained from actual determination of diazepam and its three metabolites, oxazepam, nordazepam and temazepam, in human urine after oral administration of diazepam, are also presented.     

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%.     

Increased productivity in quantitative bioanalysis using a monolithic column coupled with high-flow direct-injection liquid chromatography/tandem mass spectrometry

The feasibility of using a monolithic column as the analytical column in conjunction with high-flow direct-injection liquid chromatography/tandem mass spectrometry (LC/MS/MS) to increase productivity for quantitative bioanalysis has been investigated using plasma samples containing a drug and its epimer metabolite. Since the chosen drug and its epimer metabolite have the same selected reaction monitoring (SRM) transitions, chromatographic baseline separation of these two compounds was required. The results obtained from this monolithic column system were directly compared with the results obtained from a previously validated assay using a conventional C18 column as the analytical column. Both systems have the same sample preparation, mobile phases and MS conditions. The eluting flow rate for the monolithic column system was 3.2 mL/min (with 4:1 splitting) and for the C18 column system was 1.2 mL/min (with 3:1 splitting). The monolithic column system had a run time of 5 min and the conventional C18 column system had a run time of 10 min. The methods on the two systems were found to be equivalent in terms of accuracy, precision, sensitivity and chromatographic separation. Without sacrificing the chromatographic separation, sensitivity, accuracy and precision of the method, the reduced run time of the monolithic column method increased the sample throughput by a factor of two.     

On-line solid phase extraction using the Prospekt-2 coupled with a liquid chromatography/tandem mass spectrometer for the determination of dextromethorphan, dextrorphan and guaifenesin in human plasma

An on-line liquid chromatography/tandem mass spectrometry (LC-MS/MS) procedure, using the Prospekt- 2 system, was developed and used for the determination of the levels of the active ingredients of cough/cold medications in human plasma matrix. The experimental configuration allows direct plasma injection by performing on- line solid phase extraction (SPE) on small cartridge columns prior to elution of the analyte(s) onto the analytical column and subsequent MS/MS detection. The quantitative analysis of three analytes with differing polarities, dextromethorphan (DEX), dextrorphan (DET) and guaifenesin (GG) in human plasma presented a significant challenge. Using stable-isotope-labeled internal standards for each analyte, the Prospekt-2 on-line methodology was evaluated for sensitivity, suppression, accuracy, precision, linearity, analyst time, analysis time, cost, carryover and ease of use. The lower limit of quantitation for the on-line SPE procedure for DEX, DET and GG was 0.05, 0.05 and 5.0 ng mL(-1), respectively, using a 0.1 mL sample volume. The linear range for DEX and DET was 0.05-50 ng mL(-1) and was 5-5,000 ng mL(-1) for GG. Accuracy and precision data for five different levels of QC samples were collected over three separate days. Accuracy ranged from 90% to 112% for all three analytes, while the precision, as measured by the %RSD, ranged from 1.5% to 16.0%     

Quantification and confirmation of anionic, cationic and neutral pesticides and transformation products in water by on-line solid phase extraction-liquid chromatography-tandem mass spectrometry

Two on-line SPE-LC-ESI-MS/MS methods have been developed for the rapid determination and confirmation of 18 polar pesticides and nine transformation products (TPs) in water samples. Given the very different physico-chemical characteristics of the analytes, it was not feasible the simultaneous determination of all selected compounds in only one method. Thus, it was necessary to use heptafluorobutyric acid and formic acid in order to obtain good retention in the SPE cartridge for basic and acidic analytes, respectively. The developed analytical methodology based on the direct injection of 2 mL of water sample in the system allowed the quantification of all analytes at the 25 ng/L level (LOQ) with limits of detection normally lower than 5 ng/L. Satisfactory recoveries (70-110%) were obtained for most compounds in ground and surface water samples. Some exceptions were found mainly in surface water, due to the ion suppression produced by the higher amount of matrix interferents in these samples. The acquisition of two MS/MS transitions for each compound allowed the reliable confirmation of positive findings even at the LOQ level. The developed methodology was applied to real ground and surface water samples showing the interest of including TPs in monitoring methods, as several of them were found at concentrations higher than that of parent compounds.     

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.     

Determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, and 3,4-methylenedioxymethamphetamine in urine by online solid-phase extraction and ion-pairing liquid chromatography with detection by electrospray tandem mass spectrometry

A method using an online solid-phase extraction (SPE) and ion-pairing liquid chromatography with electrospray tandem mass spectrometry (LC/ES-MS/MS) was developed for determination of amphetamine (Amp), methamphetamine (mAmp), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA) in urine samples. A SPE cartridge column with both hydrophilic and lipophilic functions was utilized for online extraction. A reversed-phase C18 LC column was employed for LC separation and MS/MS was used for detection. Trifluoroacetic acid was added to the mobile phase as an ion-pairing reagent. This method was fully automated and the extraction and analysis procedures were controlled by a six-port switch valve. Recoveries ranging from 85-101% were measured. Good linear ranges (10-500 ng/mL) for Amp and mAmp were determined. For MDA, MDMA and MDEA, dual linear ranges were obtained from 5-100 and 100-500 ng/mL, respectively. The detection limit of each analytical compound, based on a signal-to-noise ratio of 3, ranged from 1-3 ng/mL. The applicability of this newly developed method was examined by analyzing several urine samples from drug users. Good agreement was obtained between the results from this method and a literature GC/MS method.     

Evaluation of low-pressure gas chromatography linked to ion-trap tandem mass spectrometry for the fast trace analysis of multiclass pesticide residues

A rapid multiresidue method for the analysis of 72 pesticides has been developed using a single injection with low-pressure gas chromatography/tandem mass spectrometry (LP-GC/MS/MS). The LP-GC/MS/MS method used a short capillary column of 10 m x 0.53 mm i.d. x 0.25 microm film thickness coupled with a 0.6 m x 0.10 mm i.d. restriction at the inlet end. Optimal LP-GC conditions were determined which achieved the fastest separation in MS/MS detection mode. Also MS/MS conditions were optimized in order to increase sensitivity and selectivity. The analytical parameters of the LP-GC/MS/MS method were compared with those obtained by GC/MS/MS using a conventional capillary column (30 m x 0.25 mm i.d. x 0.25 microm film thickness). Better precision and sensitivity values were obtained with the LP-GC/MS/MS approach. The limits of detection (LOD) of the compounds ranged from 0.1 to 14.1 microg L(-1) for LP-GC/MS/MS, lower than those obtained for conventional GC/MS/MS that ranged from 0.1 to 17.5 microg L(-1). The peak widths obtained with the short column in LP-GC are similar to those obtained using conventional capillary GC columns, and the peaks can be successfully identified by MS/MS detection with the conventional scan speed of ion-trap instruments. In addition, the analysis time was significantly reduced with LP-GC/MS/MS (32 min) versus GC/MS/MS (72 min), allowing the number of samples analyzed per day in a routine laboratory to be doubled.     

On-line strategies for determining trace levels of nitroaromatic explosives and related compounds in water

We report the development and tests of several systems for the simultaneous determination of 18 energetic compounds and related congeners in untreated water samples. In these systems a Restricted Access Material trap or liquid-chromatography precolumn (with a C(18) or porous graphitic carbon, PGC, stationary phase) followed by a PGC analytical column are used for sample clean-up, enrichment and separation of the trace level analytes, which are then analyzed by mass spectrometry (MS). The relative merits of two MS ionization interfaces (atmospheric pressure chemical ionization, APCI, and atmospheric pressure photoionization, APPI) were also compared for the MS identification and quantification of these analytes. APCI was found to be superior in cases where both alternatives are applicable. A major drawback when applying APPI is that no signal is obtained for the cyclic nitramines and nitrate esters. Using APCI, a wide spectrum of unstable compounds can be determined in a single analysis, and the feasibility of using large volume samples (up to 100 mL) in combination with the sensitivity of the MS detection system provide method detection limits ranging from 2.5 pg/mL (for 2,4-dinitrotoluene and 2,6-diamino-6-nitrotoluene) to 563 pg/mL (for pentaerythritol tetranitrate, PETN), with repeatability ranging from 2 to 7%. Other chemometric parameters such as robustness, selectivity, repeatability, and intermediate precision were also evaluated in the validation of the extraction methods for use in water analysis. Tests with untreated groundwater and drinking water samples, spiked with 20 ng of the analytes, yielded results similar to those obtained with high purity water samples.     

Photostability studies for micellar liquid chromatographic determination of nifedipine in serum and urine samples

Nifedipine is a photosensitive compound that is converted into its 4-(2-nitrophenyl) pyridine and 4-(2-nitrosophenyl) pyridine homologue. In order to obtain the most adequate conditions for handling nifedipine solutions in the analytical laboratory, a number of studies on the decomposition of this compound were performed. A simple micellar liquid chromatographic procedure was described to determine nifedipine in different biological matrices such as serum and urine, and to control its decomposition. To perform the analysis, nifedipine was dissolved in 0.1 m SDS at pH 3 and chromatographed using a mobile phase containing 0.125 m SDS-3% pentanol, pH 3 on a C18 column and UV detection at 235 nm. The chromatographic analysis time was 8 min. The response of the drug for both biological matrices was linear in the 1-100 microg/mL range, with r2>0.997 at all times. Repeatability, intermediate precision (CV, %) and limits of quantification and detection (ng/mL) were 0.19, 4.3, 104 and 31 in serum and 0.81, 2.1, 136 and 41 in urine. The method developed here does not show interferences or matrix effects produced by endogenous compounds. Micellar media and mobile phases have the advantage of stabilising the compounds, thus preventing photodegradation and allowing the direct injection of biological samples.     

Development and validation of an on-line two-dimensional reversed-phase liquid chromatography-tandem mass spectrometry method for the simultaneous determination of prostaglandins E(2) and F(2alpha) and 13,14-dihydro-15-keto prostaglandin F(2alpha) levels in human plasma

We developed and validated an on-line reverse-phase two-dimensional LC/MS/MS (2D-LC/MS/MS) system for simultaneous determination of the levels of prostaglandin (PG) E(2) as well as PGF(2alpha) and its metabolite 13,14-dihydro-15-keto PGF(2alpha) (F(2alpha)-M) in human plasma. Analytes were extracted by a three-step solid-phase extraction. Samples were then analyzed by on-line 2D-LC/MS/MS with electrospray ionization in negative mode. The 2D-LC system is composed of two reverse-phase analytical columns with a trapping column linking the two analytical columns. While an acidic buffer was used for both separation dimensions, differing organic solvents were employed for each dimension: methanol for the first and acetonitrile for the second to increase resolving power. The 2D-LC/MS/MS method was highly selective and sensitive with a significantly lower limit of quantitation (0.5 pg/mL for PGE(2) and 2.5 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively). Linearity of the 2D-LC/MS/MS system was demonstrated for the calibration ranges of 0.5-50 pg/mL for PGE(2) and 2.5-500 pg/mL for PGF(2alpha) and F(2alpha)-M, respectively. Acceptable precision and accuracy were obtained throughout the calibration curve ranges. This highly selective and sensitive method was successfully utilized to determine the endogenous levels of PGE(2), PGF(2alpha), and F(2alpha)-M in plasma samples from six (four male and two female) normal volunteers. The mean concentrations for each analyte were 0.755 pg/mL for PGE(2), 5.70 pg/mL for PGF(2alpha) and 9.48 pg/mL for F(2alpha)-M.     

Fully automated analysis of estrogens in environmental waters by in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry

A simple, rapid and sensitive method for the determination of five estrogens, estrone, 17beta-estradiol, estriol, ethynyl estradiol, and diethylstilbestrol, was developed using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS). These estrogens were separated within 8 min by HPLC using an XDB-C8 column and 0.01% ammonia/acetonitrile (60/40, v/v) at a flow rate of 0.2 mL/min. Electrospray ionization conditions in the negative ion mode were optimized for MS/MS detection of the estrogens. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 microL of sample using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC/MS/MS method, good linearity of the calibration curve (r > or = 0.9996) was obtained in the concentration range from 10 to 200 pg/mL for all compounds examined. The limits of detection (S/N= 3) of the five estrogens examined ranged from 2.7 to 11.7 pg/mL. The in-tube SPME method showed 34-90-fold higher sensitivity than the direct injection method (5 microL injection). This method was applied successfully to the analysis of environmental water samples without any other pretreatment and interference peaks. Several surface water and wastewater samples were collected from the area around Asahi River, and estriol was detected at 35.7 pg/mL in the effluent of a sewage treatment plant. The recoveries of estrogens spiked into river waters were above 86%, except for estriol, and the relative standard deviations were below 0.9-8.8%.     

Ultra-performance liquid chromatography/tandem mass spectrometric quantification of structurally diverse drug mixtures using an ESI-APCI multimode ionization source

We report in this paper an ultra-performance liquid chromatography/tandem mass spectrometric (UPLC(R)/MS/MS) method utilizing an ESI-APCI multimode ionization source to quantify structurally diverse analytes. Eight commercial drugs were used as test compounds. Each LC injection was completed in 1 min using a UPLC system coupled with MS/MS multiple reaction monitoring (MRM) detection. Results from three separate sets of experiments are reported. In the first set of experiments, the eight test compounds were analyzed as a single mixture. The mass spectrometer was switching rapidly among four ionization modes (ESI+, ESI-, APCI-, and APCI+) during an LC run. Approximately 8-10 data points were collected across each LC peak. This was insufficient for a quantitative analysis. In the second set of experiments, four compounds were analyzed as a single mixture. The mass spectrometer was switching rapidly among four ionization modes during an LC run. Approximately 15 data points were obtained for each LC peak. Quantification results were obtained with a limit of detection (LOD) as low as 0.01 ng/mL. For the third set of experiments, the eight test compounds were analyzed as a batch. During each LC injection, a single compound was analyzed. The mass spectrometer was detecting at a particular ionization mode during each LC injection. More than 20 data points were obtained for each LC peak. Quantification results were also obtained. This single-compound analytical method was applied to a microsomal stability test. Compared with a typical HPLC method currently used for the microsomal stability test, the injection-to-injection cycle time was reduced to 1.5 min (UPLC method) from 3.5 min (HPLC method). The microsome stability results were comparable with those obtained by traditional HPLC/MS/MS.     

Qualitative detection of diuretics and acidic metabolites of other doping agents in human urine by high-performance liquid chromatography–tandem mass spectrometry: Comparison between liquid–liquid extraction and direct injection

Direct injection of urine has gained interest in the field of analytical toxicology, including doping control analysis. However, implementation of a direct urinalysis method for the LC–MS/MS detection of 34 diuretics and 9 other doping agents yielded several analytical problems, which were not observed using a traditional liquid–liquid extraction. Therefore a comparative study was made between liquid–liquid extraction and direct injection. Comparison of validation results showed that the liquid–liquid extraction at pH 7 allows to analyze samples without major drawbacks regarding matrix effects. Hence, good sensitivity was observed and detection limits ranged between 1 and 250 ng/mL for all compounds. In the direct injection approach shifted retention times were observed for several acidic and basic compounds due to unwanted matrix effects. This shift was reduced by a 25-fold dilution of the urine samples. Besides the improved retention time stability the diluted samples also exhibited lower ion suppression than the undiluted ones. After 25-fold dilution, detection limits ranged between 10 and 250 ng/mL for all compounds. Since these detection limits are at or below the minimum required performance level, imposed by the World Anti-Doping Agency, the method could be applied to routine anti-doping analysis. Samples, previously declared positive, were reanalysed using both the liquid–liquid extraction and direct injection. With both techniques all 26 samples were found to be positive, showing the applicability of direct injection for the analysis of diuretics.     

A direct injection high-throughput liquid chromatography tandem mass spectrometry method for the determination of a new orally active alphavbeta3 antagonist in human urine and dialysate

A generic high-throughput liquid chromatography (HTLC) tandem mass spectrometry (MS/MS) assay for the determination of compound I in human urine and dialysate (hemodialysis) was developed and validated. By using the HTLC on-line extraction technique, sample pretreatment was not necessary. The sample was directly injected onto a narrow bore large particle size extraction column (50 x 1.0 mm, 60 microm) where the sample matrix was rapidly washed away using a high flow rate (5 mL/min) aqueous mobile phase while analytes were retained. The analytes were subsequently eluted from the extraction column onto an analytical column using an organic-enriched mobile phase prior to mass spectrometric detection. The analytes were then eluted from the analytical column to the mass spectrometer for the determination. The linear dynamic range was 2.0-6000 ng/mL for the urine assay and 0.1-300 ng/mL for the dialysate assay. Intraday accuracy and precision were evaluated by analyzing five replicates of calibration standards at all concentrations used to construct the standard curve. For the urine assay, the precision (RSD%, n=5) ranged from 1.9 to 8.0% and the accuracy ranged from 87.8 to 105.2% of nominal value. For the dialysate assay, the precision (RSD%, n=5) ranged from 1.1 to 10.0% and the accuracy from 94.5 to 105.2% of nominal value. In-source fragmentation of the acyl glucuronide metabolite (compound III) did not interfere with the determination of parent compound I. The developed HTLC/MS/MS methodology was specific for compound I in the presence of compound III. Column life-time is increased and sample analysis time is decreased over traditional reversed-phase methods when direct injection assays for urine and dialysate are coupled with the technology of HTLC.     

Comparison of analytical approaches for liquid chromatography/mass spectrometry determination of the alcohol biomarker ethyl glucuronide in urine

Official guidelines originating from a European Union directive regulate requirements for analytical methods used to identify chemical compounds in biological matrices. This study compared different liquid chromatography/electropray ionization mass spectrometry (LC/ESI‐MS) and tandem mass spectrometry (LC/ESI‐MS/MS) procedures for accurate determination of the conjugated ethanol metabolite and alcohol biomarker ethyl glucuronide (EtG) in urine, and the value of combined EtG and ethyl sulfate (EtS) measurement. Analysis was carried out on 482 urines following solid‐phase extraction (SPE) sample cleanup or using direct injection of a diluted sample. SPE combined with LC/MS/MS was demonstrated to be the most selective and sensitive method and was chosen as reference method. The EtG results by different methods showed good correlation (r = 0.96–0.98). When comparing five reporting limits for EtG in the range 0.10–1.00 mg/L, the overall agreement with the reference method (frequency of true positives plus true negatives) was 82–97% for direct‐injection LC/MS/MS, 90–97% for SPE‐LC/MS, 86–98% for direct‐injection LC/MS, and 86–98% for direct‐injection LC/MS analysis of EtG and EtS. Most deviations were attributable to uncertainty in quantitation, when the value was close to a cutoff but the respective results were slightly above and below, or vice versa, the critical limit. However, for direct‐injection LC/MS/MS, despite earning 4 identification points, equally many negative results were due to a product ion ratio outside the ±20% deviation accepted by the guidelines. These results indicate that the likelihood of different analytical methods to provide reliable analytical results depends on the reporting limit applied. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of MK-0431 in human plasma using high turbulence liquid chromatography online extraction and tandem mass spectrometry

A robust and sensitive method using high turbulence liquid chromatography (HTLC) online extraction with tandem mass spectrometry (MS/MS) for the determination of MK-0431 in human plasma was developed and validated to support the clinical studies. This HTLC online extraction method eliminated the time-consuming offline sample extraction procedures and significantly increased productivity. A narrow bore large particle size reversed-phase column (Cyclone, 50 x 1.0 mm, 60 microm) and a BDS Hypersil C18 column (30 x 2.1 mm, 3 microm) were used as extraction and analytical columns, respectively. The linear dynamic range of the calibration curve was 0.5 to 1000 ng/mL. Intraday validation was conducted using five calibration curves prepared in five lots of human control plasma, and the intraday precision (RSD%) was from 2.4 to 9.0% and the accuracy was from 98.0 to 103% of the nominal value. The intraday precision (RSD%, n = 5) for plasma quality control (QC) samples varied from 2.0 to 5.3% and accuracy from 103 to 105% of the nominal value. The interday precision (RSD%) for 100 sets of plasma QC samples in 29 analytical runs varied from 6.3 to 9.0% and the accuracy from 98.8 to 104% of the nominal value. No significant difference was observed between the interday and intraday precision and accuracy of the QC samples.     

'LC-electrolyte effects' improve the bioanalytical performance of liquid chromatography/tandem mass spectrometric assays in supporting pharmacokinetic study for drug discovery

The development of rapid and sensitive bioanalytical methods in a short time frame with acceptable levels of precision and accuracy is imperative for successful drug discovery. We previously reported that the use of a mobile phase containing an extremely low concentration of ammonium formate or formic acid increased analyte electrospray ionization (ESI) response and controlled against matrix effects. We designated these favorable effects 'LC-electrolyte effects'. In order to support rapid pharmacokinetic (PK) studies for drug discovery, we applied LC-electrolyte effects to the development of generic procedures that can be used to quickly generate reliable PK data for compound candidates. We herein demonstrate our approach using four model tested compounds (Compd-A, -B, -C, and -D). The analytical methods involve generic protein precipitation for sample clean-up, followed by application of fast liquid chromatographic (LC) gradients and the subsequent use of electrospray ionization tandem mass spectrometry (ESI-MS/MS) for individual measurement of the tested compounds in 20-microL plasma samples. Good linearity over the concentration range of 1.6 or 8-25000 ng/mL (r(2) > 0.99), precision (RSD, 0.45-13.1%), and accuracy (91-112%) were achieved through the use of a low dose of formic acid (0.4 mM or 0.015 per thousand) in the methanol/water-based LC mobile phase. The analytical method was quite sensitive, providing a lower limit of quantification of 1.6 pg on-column except for Compd-C (8 pg), and showed negligible ion suppression caused by matrix components. Finally, the assay suitability was demonstrated in simulated discovery PK studies of the tested compounds with i.v./p.o. dosing of rats. This new assay approach has been adopted with good results in our laboratory for many recent discovery PK studies.