Liquid chromatographic-electrospray tandem mass spectrometric determination of clarithromycin in human plasma.

A rapid, sensitive and specific LC-MS-MS method has been developed for the determination of clarithromycin (CLA) in human plasma using roxithromycin (ROX) as the internal standard. Samples were prepared via liquid-liquid extraction with methyl tert-butyl ether (MTBE) and chromatographed on a Supelco RP(18) (4.6 x 50 mm, 3 microm particle size) column with a mobile phase consisting of acetonitrile:methanol:60 mM (pH 3.5) ammonium acetate buffer (32.5:32.5:35) at a constant flow rate of 0.8 mL/min. The run time was 3 min with retention times of approximately 1.65 and 1.70 min for CLA and ROX, respectively. Detection was performed on a PE Sciex API 365 mass spectrometer equipped with a turboionspray ionization source in multiple reaction monitoring (MRM) mode. The MRM pairs were m/z 748.5 --> m/z 158.2 for CLA and m/z 837.7 --> m/z 679.3 for ROX, respectively, with dwell times of 200 ms for each transition. The validated calibration curve range was 5.00-5000 ng/mL, based on 0.100 mL plasma sample volume with signal-to-noise ratio (S/N) greater than 60 for CLA at the lower limit of quantification level (5.00 ng/mL). The correlation coefficients (r(2)) of the calibration curves were better than or equal to 0.996. The inter-day (n = 18) precision and accuracy of the quality control (QC) samples were less than 3.58% RSD (relative standard deviation) and -10.8% bias, respectively. The intra-day (n = 6) precision and accuracy of the quality control samples were less than 5.0 and 12.6%, respectively. There was no significant deviation from the nominal values after a 10-fold dilution of high concentration QC samples using blank matrix. The QC samples were stable when left on the bench for 24 h or after three freeze-thaw cycles. The processed samples were also stable in HPLC autosampler at 10C for over 72 h. No matrix ionization suppression was observed when extracted blank matrix or reconstitution solvent was injected onto the system with post-column infusion of clarithromycin and roxithromycin. No carryover was observed when an extracted blank plasma sample was injected immediately after a 5000 ng/mL ULOQ (the upper limit of quantification) standard. The mean recovery was 81.5 and 78.3%, respectively, for clarithromycin and internal standard.

Determination of S-phenylmercapturic acid in human urine using an automated sample extraction and fast liquid chromatography-tandem mass spectrometric method.

S-phenylmercapturic acid is widely accepted as a specific biomarker for the evaluation of benzene exposure. Here, we describe a fast, specific and sensitive high-performance liquid achromatography coupled with tandem mass spectrometry (LC-MS/MS) method that has been developed and validated for the determination of S-phenylmercapturic acid in human urine. Isotope-labeled S-phenylmercapturic acid-d5 was used as internal standard to improve the method ruggedness. The fully automated solid-phase extraction method on a 96-well Oasis MAX (mix-mode anion exchange) plate was employed to clean up the urine samples before analysis. The rapid LC-MS/MS analysis of extracted samples was achieved on a Genesis C18 column with a run time of only 3 min. Negative electrospray ionization with multiple reaction monitoring (ESI-MRM) mode was used to detect S-phenylmercapturic acid (m/z 238 --> 109) and S-phenylmercapturic acid -d5 (m/z 243 --> 114). The method fulfils all the standard requirements of method validation. The calibration curve was linear within the concentration range 0.400-200 ng/mL. The method performed accurately and precisely in validation with <7.5% relative error and <6.5% relative standard deviation of quality control samples. The method efficacy was also verified by the analysis of urine samples from 12 smokers and 12 non-smokers. With the fully automated sample cleanup procedure and the fast LC-MS/MS analysis, a sample analysis throughput of 384 samples per day could be achieved.

Analysis of omeprazole and 5-OH omeprazole in human plasma using hydrophilic interaction chromatography with tandem mass spectrometry (HILIC-MS/MS)--eliminating evaporation and reconstitution steps in 96-well liquid/liquid extraction.

Bioanalytical methods using liquid/liquid extraction (LLE) and liquid chromatography with electrospray tandem mass spectrometry (LC-MS/MS) are widely used. The organic extracts need to be evaporated and reconstituted, hampering further improvement of throughput and automation. In this study, we demonstrated a novel approach of eliminating these two steps in 96-well LLE by using hydrophilic interaction chromatography with MS/MS (HILIC-MS/MS) on silica column with high organic/low aqueous mobile phase. Omeprazole, its metabolite 5-OH omeprazole, and internal standard desoxyomeprazole, were extracted from 0.05 ml of human plasma using 0.5 ml of ethyl acetate in a 96-well plate. A portion (0.1 ml) of the ethyl acetate extract was diluted with 0.4 ml of acetonitrile and 10 microl was injected onto a Betasil silica column (50 mm x 3.0 mm, 5 microm) and detected by API 3000 and 4000 with (+) ESI. Mobile phase with linear gradient elution consists of acetonitrile, water, and formic acid (from 95:5:0.1 to 73.5:26.5:0.1 in 2 min). The flow rate was 1.5 ml/min with total run time of 2.75 min. The method was validated for a low limit of quantitation at 2.5 ng/ml for both analytes. The method was also validated for specificity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <4.4% relative standard deviation (R.S.D.) and 4.1% relative error (R.E.) for omeprazole, and 4.5% R.S.D. and 5.6% R.E. for 5-OH omeprazole, respectively.

A novel approach to perform metabolite screening during the quantitative LC-MS/MS analyses of in vitro metabolic stability samples using a hybrid triple-quadrupole linear ion trap mass spectrometer.

In vitro metabolic stability experiments using microsomes or other liver preparations are important components in the discovery and lead-optimization stages of compound selection in the pharmaceutical industry. Currently, liquid chromatography-tandem mass spectrometric (LC-MS/MS) support of in vitro metabolic stability studies primarily involves the monitoring of disappearance of parent compounds, using selected reaction monitoring (SRM) on triple-quadrupole instruments. If moderate to high turnover is observed, separate metabolite identification experiments are then conducted to characterize the biotransformation products. In this paper, we present a novel method to simultaneously perform metabolite screening in addition to the quantitative stability measurements, both within the same chromatographic run. This is accomplished by combining SRM and SRM-triggered, information-dependent acquisition (IDA) of MS/MS spectra on a hybrid triple-quadrupole linear ion trap (QqQLIT) mass spectrometer. Microsomal stability experiments using model compounds, bufuralol, propranolol, imipramine, midazolam, verapamil and diclofenac, were used to demonstrate the applicability of our approach. This SRM + SRM-IDA approach generated metabolic stability results similar to those obtained by conventional SRM-only approach. In addition, MS/MS spectra from potential metabolites were obtained with the enhanced product ion (EPI) scan function of LIT during the same injection. These spectra were correlated to the spectra of parent compounds to confirm the postulated structures. The time-concentration profiles of identified metabolites were also estimated from the acquired data. This approach has been successfully used to support discovery programs.

Simple means to alleviate sensitivity loss by trifluoroacetic acid (TFA) mobile phases in the hydrophilic interaction chromatography-electrospray tandem mass spectrometric (HILIC-ESI/MS/MS) bioanalysis of basic compounds.

Trifluoroacetic acid (TFA) is a commonly used additive in HPLC and LC-MS analysis of basic compounds. It is also routinely added to aqueous-organic mobile phases utilized in the hydrophilic interaction chromatography-electrospray tandem mass spectrometry (HILIC-ESI/MS/MS) technique used in our laboratories for bioanalysis. However, TFA is known to suppress the ESI signals of analytes due to its ability to form gas-phase ion pairs with positively-charged analyte ions. The most common method to overcome this problem involves the post-column addition of a mixture of propionic acid and isopropanol. However the post-column addition setup requires additional pumps and is not desirable for continuous analysis of large amounts of samples. In this paper we present a simple yet very effective means of minimizing the negative effect of TFA in bioanalysis by direct addition of 0.5% acetic acid or 1% propionic acid to mobile phases containing either 0.025 or 0.05% TFA. A factor of two- to five-fold signal enhancement was achieved for eight basic compounds studied. Furthermore, chromatography integrity was maintained even with the addition of acetic acid and propionic acid to existing TFA mobile phases. This method has been successfully applied to the HILIC-ESI/MS/MS high-throughput analysis of extracted biological samples to support pre-clinical and clinical studies.

Simultaneous determination of norethindrone and ethinyl estradiol in human plasma by high performance liquid chromatography with tandem mass spectrometry--experiences on developing a highly selective method using derivatization reagent for enhancing sensitivity.

In the present work, for the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous analysis of norethindrone, and ethinyl estradiol, was developed and validated over the concentration range of 50-10000pg/ml and 2.5-500pg/ml, respectively, using 0.5 ml of plasma sample. Norethindrone, ethinyl estradiol, and their internal standards norethindrone-(13)C2, and ethinyl estradiol-d4, were extracted from human plasma matrix with n-butyl chloride. After evaporation of the organic solvent, the extract was derivatized with dansyl chloride and the mixture was injected onto the LC-MS/MS system. The gradient chromatographic elution was achieved on a Genesis RP-18 (50 mm x 4.6 mm, 3 microm) column with mobile phase consisted of acetonitrile, water and formic acid. The flow rate was 1.0 ml/min and the total run time was 5.0 min. Important parameters such as sensitivity, linearity, matrix effect, reproducibility, stability, carry-over and recovery were investigated during the validation. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <6.8% relative standard deviation (RSD) and 4.4% relative error (RE) for norethindrone, and 4.2% RSD and 5.9% RE for ethinyl estradiol, respectively. Chromatographic conditions were optimized to separate analytes of interest from the potential interference peaks, arising from the derivatization. This method could be used for pharmacokinetic and drug-drug interaction studies in human subjects.

The use of chemical derivatization to enhance liquid chromatography/tandem mass spectrometric determination of 1-hydroxypyrene, a biomarker for polycyclic aromatic hydrocarbons in human urine.

This article presents an analytical approach that used chemical derivatization to enhance mass spectrometric (MS) response in electrospray ionization (ESI) mode of 1-hydroxypyrene (1-OHP), a commonly used biomarker to monitor human exposure to polycyclic aromatic hydrocarbons (PAHs). The enhancement successfully enabled the desired detection of 50 pg/mL in human urine. The introduction of an MS-friendly dansyl group to 1-OHP enhanced both ionization efficiency in the ESI source and collision-activated dissociation (CAD) in the collision cell. The response increase was estimated to be at least 200-fold, and enabled the reduction of sample size to only 100 microL. The selective MS detection also facilitated a fast (run time 3 min) liquid chromatography (LC) method which successfully resolved the analyte and interferences. The sample processing procedure included enzymatic hydrolysis of glucuronide and sulfate conjugates, liquid-liquid extraction, derivatization with dansyl chloride and a final liquid-liquid extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (50-1000 pg/mL), accurate and precise for the quantitation of 1-OHP in human urine. This is the first report of using chemical derivatization to enhance MS/MS detection with fast chromatography in the determination of 1-OHP in human urine.

High-throughput biological sample analysis using on-line turbulent flow extraction combined with monolithic column liquid chromatography/tandem mass spectrometry.

A high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method, which combines on-line sample extraction through turbulent flow chromatography with a monolithic column separation, has been developed for direct injection analysis of drugs and metabolites in human plasma samples. By coupling a monolithic column into the system as the analytical column, the method enables running 'dual-column' extraction and chromatography at higher flow rates, thus significantly reducing the time required for the transfer and mixing of extracted fraction onto the separation column as well as the time for gradient separation. A strategy of assessing and reducing the matrix suppression effect on the on-line extraction LC/MS/MS has also been discussed. Experiments for evaluating the resolution, peak shape, sensitivity, speed, and matrix effect were conducted with dextromethorphan and its metabolite dextrorphan as model compounds in human plasma matrix. It was demonstrated that the total run time for this assay with a baseline separation of two analytes is less than 1.5 min.

Hydrophilic interaction liquid chromatographic tandem mass spectrometric determination of atenolol in human plasma.

A hydrophilic interaction liquid chromatographic method with tandem mass spectrometry for the determination of atenolol, a beta-blocking agent, in human plasma has been developed and validated over the curve range of 10--2000 ng/mL. The assay was based on protein precipitation followed by evaporation of the extraction solvent, reconstitution with acetonitrile, and chromatography on an Hypersil silica column (50 x 4.6 mm) using a low aqueous--high organic mobile phase. The mobile phase consists of 85% acetonitrile, 15% water, 0.5% acetic acid and 0.04% trifluoroacetic acid and runs isocratically at a flow rate of 2.0 mL/min. The column ef fluent was split so that 50% of it was transferred into the LC-MS/MS interface operated in positive electrospray ionization mode. The chromatographic run time was 2.0 min per injection. Atenolol and the internal standard, atenolol-d(7), showed a retention time of 1.0 min. The inter-day and intra-day precision and accuracy of the quality control samples were <5.3% relative standard deviation and <8.0% relative error, respectively. To explore the application of the current method for the analysis of other beta-blocking agents, propranolol and metoprolol were tested under the same chromatographic conditions with retention times of 0.68 and 0.75 min, respectively. The present method could be used for therapeutic drug monitoring, pharmacokinetic and drug--drug interaction studies of beta-blocking agents.

Automated 96-well solid phase extraction and hydrophilic interaction liquid chromatography-tandem mass spectrometric method for the analysis of cetirizine (ZYRTEC) in human plasma--with emphasis on method ruggedness.

A high-throughput bioanalytical method based on automated sample transfer, automated solid phase extraction, and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) analysis, has been developed for the determination of cetirizine, a selective H(1)-receptor antagonist. Deuterated cetirizine (cetirizine-d(8)) was synthesized as described and was used as the internal standard. Samples were transferred into 96-well plates using an automated sample handling system. Automated solid phase extraction was carried out using a 96-channel programmable liquid-handling workstation. Solid phase extraction 96-well plate on polymer sorbent (Strata X) was used to extract the analyte. The extracted samples were injected onto a Betasil silica column (50 x 3, 5 microm) using a mobile phase of acetonitrile-water-acetic acid-trifluroacetic acid (93:7:1:0.025, v/v/v/v) at a flow rate of 0.5 ml/min. The chromatographic run time is 2.0 min per injection, with retention time of cetirizine and cetirizine-d(8) both at 1.1 min. The system consisted of a Shimadzu HPLC system and a PE Sciex API 3000 or API 4000 tandem mass spectrometer with (+) ESI. The method has been validated over the concentration range of 1.00-1000 ng/ml cetirizine in human plasma, based on a 0.10-ml sample size. The inter-day precision and accuracy of the quality control (QC) samples demonstrated <3.0% relative standard deviation (R.S.D.) and <6.0% relative error (RE). Stability of cetirizine in stock solution, in plasma, and in reconstitution solution was established. The absolute extraction recovery was 85.8%, 84.5%, and 88.0% at 3, 40, and 800 ng/ml, respectively. The recovery for the internal standard was 84.1%. No adverse matrix effects were noticed for this assay. The automation of the sample preparation steps not only increased the analysis throughput, but also increased method ruggedness. The use of a stable isotope-labeled internal standard further improved the method ruggedness. Practical issues of analyzing incurred samples were discussed. This HILIC-MS/MS method for analysis of citirizine in human plasma was successfully used to support clinical studies.

Development, validation and transfer of a hydrophilic interaction liquid chromatography/tandem mass spectrometric method for the analysis of the tobacco-specific nitrosamine metabolite NNAL in human plasma at low picogram per milliliter concentrations.

A highly sensitive bioanalytical method based on a simple liquid/liquid extraction and hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC/MS/MS) analysis has been developed, validated and transferred for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a tobacco-specific nitrosamine metabolite. Deuterated NNAL (NNAL-d(4)) was synthesized and used as the internal standard. This method can be used for the analysis of free and total NNAL (free NNAL plus NNAL-gluc) in K(3)-EDTA human plasma. Free NNAL and NNAL-d(4) are extracted from human plasma by liquid/liquid extraction. To analyze for total NNAL and the internal standard, a separate aliquot of the K(3)-EDTA human plasma is treated with beta-glucuronidase to deconjugate the NNAL-gluc; the total NNAL and internal standard are then extracted using liquid/liquid extraction. After drying down under nitrogen, the residue is reconstituted with acetonitrile and analyzed using positive ion electrospray and HILIC/MS/MS at a flow rate of 1.0 mL/min. The chromatographic run time is 1.0 min per injection, with retention time for both NNAL and NNAL-d(4) of 0.75 min with a capacity factor (k') of 2. The standard curve range for this assay is from 5.00-1000 pg/mL for both free and total NNAL, using a total plasma sample volume of 1.0 mL. The interday precision and accuracy of the quality control (QC) samples demonstrated <7.6% relative standard deviation (RSD) and <3.3% relative error (RE) for free NNAL. For total NNAL, the interday precision and accuracy of the QC samples demonstrated <11.7% RSD and <2.8% RE. Optimization of enzyme hydrolysis of NNAL-gluc is discussed in detail. The overall recoveries for free and total NNAL and IS were 68.2 and 71.5% (free) and 70.7 and 65.5% (total). No adverse matrix effects were noticed for this assay.

Direct injection of solid-phase extraction eluents onto silica columns for the analysis of polar compounds isoniazid and cetirizine in plasma using hydrophilic interaction chromatography with tandem mass spectrometry.

Isoniazid and cetirizine do not retain well on reversed-phase columns due to their high polarity. Silica columns, when operated under hydrophilic interaction conditions, do provide excellent retention of these compounds. We have developed simple and proof of concept analytical methods for the analysis of isoniazid and cetirizine in animal and human plasma, respectively. Both methods employed the approach of direct injection of solid-phase extraction (SPE) organic eluents onto silica columns for analysis, thus eliminating evaporation and reconstitution steps that are typically needed for reversed-phase liquid chromatographic analysis. Isoniazid was extracted from animal plasma samples using a Waters Oasis HLB 96-well plate and then eluted with acetonitrile, while cetirizine was extracted from human plasma with a Waters MCX mu-Elute plate and then eluted with acetonitrile containing 5% concentrated ammonium hydroxide. The direct injection of the SPE eluent onto the analytical column was necessary since significant loss of isoniazid was found during the evaporation and reconstitution steps. The method for isoniazid also enabled ultra-fast analysis due to the relatively low back-pressure exhibited by silica columns even under high flow conditions. Both methods show good linearity, accuracy and precision covering the range of 10-2000 ng/mL of isoniazid, and 1-1000 ng/mL of cetirizine in plasma. Substantial time savings were realized as a result of both the elimination of the evaporation and reconstitution steps and the fast chromatographic analysis.

Development and validation of a high-sensitivity liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with chemical derivatization for the determination of ethinyl estradiol in human plasma.

An ultra-sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the analysis of oral contraceptive ethinyl estradiol (EE) was developed and validated over the curve range of 2.5-500 pg/mL using 1 mL of human plasma sample. Ethinyl estradiol and the internal standard, ethinyl estradiol tetra-deuterated (EE-d4), were extracted from the plasma matrix with methyl t-butyl ether, derivatized with dansyl chloride and then back-extracted into hexane. The hexane phase was evaporated to dryness, reconstituted and injected onto the LC/MS/MS system. The chromatographic separation was achieved on a Luna C(18) column (50 x 2 mm, 5 micro m) with an isocratic mobile phase of 20:80 (v/v) water:acetonitrile with 1% formic acid. The offline derivatization procedure introduced the easily ionizable tertiary amine function group to EE. This greatly improved analyte sensitivity in electrospray ionization and enabled us to achieve the desired lower limit of quantitation at 2.5 pg/mL. This high sensitivity method can be used for therapeutic drug monitoring or supporting bio-equivalence and drug-drug interaction studies in human subjects.

Development and validation of a hydrophilic interaction liquid chromatography-tandem mass spectrometric method for the analysis of paroxetine in human plasma.

A sensitive, simple, fast and rugged hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the determination of paroxetine was developed and validated over curve range 0.050-50 ng/mL using only 0.4 mL plasma. This is the first published LC-MS/MS method and the low limit of quantitation of this method is 10-fold lower than previously published methods. A simple liquid-liquid extraction method using methyl-tert butyl ether (MTBE) as the extraction solvent was used to extract paroxetine and the internal standard (IS) fentanyl-d(5) from plasma. The extract was evaporated to dryness, reconstituted and injected onto a silica column using a low aqueous-high organic mobile phase. The chromatographic run time was 2.0 min per injection, with retention times of 1.1 and 1.2 min for paroxetine and IS, respectively. The detection was by monitoring paroxetine at m/z 330 --> 192 and IS at m/z 342 --> 188, respectively. The inter-day precision and accuracy of the quality control (QC) samples were <5.0% relative standard deviation (RSD) and <2.9% relative error (RE). This method can be used for supporting therapeutical drug monitoring and pharmacokinetic or drug-drug interaction studies.

Bioanalytical liquid chromatography tandem mass spectrometry methods on underivatized silica columns with aqueous/organic mobile phases.

This review article summarizes the recent progress on bioanalytical LC-MS/MS methods using underivatized silica columns and aqueous/organic mobile phases. Various types of polar analytes were extracted by using protein precipitation (PP), liquid/liquid extraction (LLE) or solid-phase extraction (SPE) and were then analyzed using LC-MS/MS on the silica columns. Use of silica columns and aqueous/organic mobile phases could significantly enhance LC-MS/MS method sensitivity, due to the high organic content in the mobile phase. Thanks to the very low backpressure generated from the silica column with low aqueous/high organic mobile phases, LC-MS/MS methods at high flow rates are feasible, resulting in significant timesaving. Because organic solvents have weaker eluting strength than water, direct injection of the organic solvent extracts from the reversed-phase solid-phase extraction onto the silica column was possible. Gradient elution on the silica columns using aqueous/organic mobile phases was also demonstrated. Contrary to what is commonly perceived, the silica column demonstrated superior column stability. This technology can be a valuable supplement to the reversed-phase LC-MS/MS.

A sensitive LC/MS/MS method using silica column and aqueous-organic mobile phase for the analysis of loratadine and descarboethoxy-loratadine in human plasma.

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) was developed and validated for the simultaneous analysis of antihistamine drug loratadine (LOR) and its active metabolite descarboethoxy-loratadine (DCL) in human plasma. Deuterated analytes, i.e. LOR-d(3) and DCL-d(3) were used as the internal standards (I.S.). Analytes were extracted from alkalized human plasma by liquid/liquid extraction using hexane. The extract was evaporated to dryness under nitrogen, reconstituted with 0.1% (v/v) of trifluoroacetic acid (TFA) in acetonitrile, and injected onto a 50 x 3.0 mm I.D. 5 microm, silica column with an aqueous-organic mobile phase consisted of acetonitrile, water, and TFA (90:10:0.1, v/v/v). The chromatographic run time was 3.0 min per injection and flow rate was 0.5 ml/min. The retention time was 1.2 and 2.0 min for LOR and DCL, respectively. The tandem mass spectrometric detection was by monitoring singly charged precursor-->product ion transitions: 383-->337 (m/z) for LOR, 311-->259 (m/z) for DCL, 388-->342 (m/z) for LOR-d(3), and 316-->262 (m/z) for DCL-d(3). The low limit of quantitation (LLOQ) was 10 pg/ml for LOR and 25 pg/ml for DCL. The inter-day precision of the quality control (QC) samples was 3.5-9.4% relative standard deviation (R.S.D.). The inter-day accuracy of the QC samples was 99.0-107.9% of the nominal values.

Assay and purity control of tetracycline, chlortetracycline and oxytetracycline in animal feeds and premixes by TLC densitometry with fluorescence detection.

Methods using TLC densitometry with fluorescence detection are described for the assay and purity control of tetracycline (TC), chlortetracycline (CTC), and oxytetracycline (OTC) in animal feeds and premixes. With a silica gel layer previously sprayed with 10% (m/v) sodium EDTA solution adjusted to pH 8.0 or 9.0, all the major impurities were separated from the main components and from each other. The mobile phase consisted of dichloromethane, methanol, and water. After development, the plate was dipped in a 30% (v/v) solution of liquid paraffin in hexane. Quantitation was realized by fluorescence densitometry at 400 nm. The limit of quantitation (LOQ) for tetracycline impurities is 0.8 microg/g, corresponding to 0.2% of the label claimed tetracycline (400 microg/g). The LOQ for impurities of tetracycline and chlortetracycline in premixes is 0.2% of the label-claimed TC (40 mg/g) and CTC (200 or 400 mg/g). The LOQ for impurities of oxytetracycline in a premix is 0.1% of the label claimed OTC (100 mg/g).

Liquid/liquid extraction using 96-well plate format in conjunction with hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the analysis of fluconazole in human plasma.

A bioanalytical method using automated sample transferring, automated liquid/liquid extraction (LLE) and hydrophilic interaction liquid chromatography-tandem mass spectrometry was developed for the determination of fluconazole in human plasma. Samples of 0.05 ml were transferred into 96-well plate using automatic liquid handler (Multiprobe II). Automated LLE was carried out on a 96-channel programmable liquid handling workstation (Quadra 96) using methyl-tetra butyl ether as the extraction solvent. The extract was evaporated to dryness, reconstituted, and injected onto a silica column using an aqueous-organic mobile phase. The chromatographic run time was 2.0 min per injection, with retention times of 1.47 and 1.44 min for fluconazole and internal standard (IS) ritonavir, respectively. The detection was by monitoring fluconazole at m/z 307-->238 and IS at m/z 721-->296, respectively. The standard curve range was 0.5-100 ng ml(-1). The inter-day precision and accuracy of the quality control samples were <7.1% relative standard deviation and <2.2% relative error.

Post-column infusion study of the 'dosing vehicle effect' in the liquid chromatography/tandem mass spectrometric analysis of discovery pharmacokinetic samples.

It has become increasingly popular in drug development to conduct discovery pharmacokinetic (PK) studies in order to evaluate important PK parameters of new chemical entities (NCEs) early in the discovery process. In these studies, dosing vehicles are typically employed in high concentrations to dissolve the test compounds in dose formulations. This can pose significant problems for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis of incurred samples due to potential signal suppression of the analytes caused by the vehicles. In this paper, model test compounds in rat plasma were analyzed using a generic fast gradient LC/MS/MS method. Commonly used dosing vehicles, including poly(ethylene glycol) 400 (PEG 400), polysorbate 80 (Tween 80), hydroxypropyl beta-cyclodextrin, and N,N-dimethylacetamide, were fortified into rat plasma at 5 mg/mL before extraction. Their effects on the sample analysis results were evaluated by the method of post-column infusion. Results thus obtained indicated that polymeric vehicles such as PEG 400 and Tween 80 caused significant suppression (> 50%, compared with results obtained from plasma samples free from vehicles) to certain analytes, when minimum sample cleanup was used and the analytes happened to co-elute with the vehicles. Effective means to minimize this 'dosing vehicle effect' included better chromatographic separations, better sample cleanup, and alternative ionization methods. Finally, a real-world example is given to illustrate the suppression problem posed by high levels of PEG 400 in sample analysis, and to discuss steps taken in overcoming the problem. A simple but effective means of identifying a 'dosing vehicle effect' is also proposed.