High-performance liquid chromatography assay for the determination of the HIV-protease inhibitor tipranavir in human plasma in combination with nine other antiretroviral medications

An accurate, sensitive and simple reverse-phase (RP) high-performance liquid chromatography (HPLC) assay has been developed and validated for the simultaneous quantitative determination of tipranavir with nine other antiretroviral drugs in plasma. A liquid-liquid extraction of the drugs in tert-butylmethylether (TBME) from 200 microL of plasma is followed by a reversed phase gradient HPLC assay with UV detection at 210 nm. The standard curve for the drug was linear in the range of 80-80,000 ng/mL for tipranavir; 10-10,000 ng/mL for nevirapine, indinavir, efavirenz, and saquinavir; and 25-10,000 ng/mL for amprenavir, atazanavir, ritonavir, lopinavir, and nelfinavir. The regression coefficient (r(2)) was greater than 0.998 for all analytes. This method has been fully validated and shown to be specific, accurate and precise. Due to an excellent extraction procedure giving good recovery and a clean baseline, this method is simple, rapid, accurate and provides excellent resolution and peak shape for all analytes. Thus this method is very suitable for therapeutic drug monitoring.     

Separation of positional isomers of nine 2‐phenethylamine‐derived designer drugs by liquid chromatography–tandem mass spectrometry

The synthesis of positional isomers of designer drugs is a common way of bypassing legal restrictions. For forensic case work, and especially for the legal assessment of cases, there is a need for screening methods capable of the unequivocal identification of positional isomers. The presented liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) method facilitates separation of positional isomers of 9 2‐phenethylamine‐derived designer drugs in different matrices including seized materials, hair, serum, and urine specimens. Chromatographic separation was achieved on a biphenyl phase using gradient elution with a total runtime of 26 minutes. The limit of detection was 25 pg/mg for hair samples and ranged from 0.1 ng/mL to 0.5 ng/mL for serum and from 0.2 ng/mL to 1.2 ng/mL for urine samples. The method proved to be selective and sensitive and showed good chromatographic resolution (R ≥ 1.2). The method was successfully applied to routine case samples.     

Research on accurate quantitation technique using liquid chromatography coupled with triple quadrupole mass spectrometry and experiment optimization—take a novel diuretic as an example

Liquid chromatography coupled with the triple quadrupole mass spectrometry (LC-QQQ MS) technique is the most commonly used technique for quantitative analysis. It is widely used in pharmacology, targeted metabolomics, Chinese medicine quality control, and other research fields. The technique monitors only characteristic precursors and product ions through multiple reaction monitoring (MRM) mode and can detect targeted molecules in complex matrix with high specificity and sensitivity. In the present study, a diarylamide derivative diuretic was used as an example to introduce the method establishment and parameter optimization of this liquid chromatography-mass spectrometry technique. Diuretic and its internal standard could be completely separated within a 5-min gradient, and the quantitative linear range was 1–1000 ng/mL with R² = 0.9996 in practical samples. This study showed that the key to the method development for LC-QQQ MS was the selection of the LC mobile phase, the elution gradient, the declustering potential, and the collision energy of compounds in MS.     

Rapid determination of nine barbiturates in human whole blood by liquid chromatography‐tandem mass spectrometry

A rapid, simple and sensitive liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed for the qualitative and quantitative analysis of nine barbiturates (barbital, phenobarbital, pentobarbital, amobarbital, secobarbital, thiopental, butalbital, butabarbital, and hexobarbital) in human whole blood. Barbiturates were extracted from 100 μL of human whole blood samples using a simple liquid‐liquid extraction (LLE) procedure, and detected by LC‐MS/MS. An UPLC C18 (2.1 mm × 100 mm, 1.7 µm) column was used at 40 °C for the separation and acetonitrile/water system was used as the mobile phase with gradient elution. This method showed excellent accuracy (86–111%) and precision (relative standard deviation <15%). The limits of detection (LODs) were 0.2 ng/mL for barbital and secobarbital and 0.5 ng/mL for the other barbiturates. The linearity ranged from 2 ng/mL to 2000 ng/mL, with r² > 0.99 over the range. This method achieved the separation and detection of pentobarbital and amobarbital at the same time in a convenient way. Moreover, it was both simple and sensitive for the determination of nine most commonly used barbiturate drugs, which was meaningful in the field of clinical and forensic toxicology. Copyright © 2016 John Wiley & Sons, Ltd.     

Pharmacokinetic monitoring of HIV-1 protease inhibitors in the antiretroviral therapy

Combination therapy with protease inhibitors (PIs) is used for the treatment of patients infected with the human immunodeficiency virus (HIV). To achieve optimal drug concentrations for viral suppression and avoidance of drug toxicity, therapeutic drug monitoring of PIs levels has been considered essential. In this study an analytical procedure for simultaneous monitoring the plasma concentrations of a total four protease inhibitors: indinavir, lopinavir, nelfinavir and saquinavir was presented. The plasma samples were liquid/liquid extracted and subjected to high performance liquid chromatography (HPLC) analysis. The lopinavir and saquinavir in the patient plasma samples were monitored by ultraviolet detection at 215 nm. Extraction procedure using methyl tert-butyl ether and the mobile phase consisted from acetonitrile with phosphate buffer on a Symmetry C18 column were used to monitor these two compounds. Linearity of the method was obtained in the concentration range of 0.1 - 15.0 mg/mL for all four protease inhibitors. Under steady state conditions, the plasma concentrations of protein inhibitors in 23 patients were determined and area under the plasma concentration-time curve was estimated to maintain optimal viral suppression. We developed a simple and specific method for the simultaneous determination of lopinavir and saquinavir.     

Liquid chromatography-tandem mass spectrometry method for the simultaneous analysis of multiple hallucinogens, chlorpheniramine, ketamine, ritalinic acid, and metabolites, in urine

A validated method for the simultaneous analysis of multiple hallucinogens, chlorpheniramine, ketamine, ritalinic acid, and several metabolites is presented. The procedure comprises a sample clean-up step, using mixed-mode solid-phase extraction followed by liquid chromatography (LC)-tandem mass spectrometry analysis. Chromatographic separation was achieved using a Sunfire C(8) column eluted with a mixture of formate buffer, methanol, and acetonitrile. The applied LC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time and two precursor-product ion transitions for the non-deuterated analogues. Validation of the method was performed using 500 microL of urine. The limits of quantification (LOQ) for LSD and 2-oxo-3-hydroxy-LSD were 0.05 and 1 ng/mL, respectively, and ranged, for the other hallucinogens, from 0.5 to 10 ng/mL. Linear and quadratic regression was observed from the LOQ of each compound to 12.5 ng/mL for LSD, 50 ng/mL for 2-oxo-3-hydroxy-LSD and 500 ng/mL for the others (r(2) > 0.99). Precision for the QC samples, spiked at a minimum of two concentrations, was calculated [%CV and %bias < 20% for most of the compounds, except for bufotenine and cathinone (%bias < 24%), and ibogaine (%bias < 30%)]. Extraction was found to be both reproducible and efficient with recoveries > 87% for all the analytes. Furthermore, the processed samples were demonstrated to be stable in the autosampler for at least 24 h. Finally, the validated method was applied to the determination of chlorpheniramine, ketamine, LSD, and psilocin in authentic urine samples.     

Quantification of tricyclic antidepressants and monoamine oxidase inhibitors by high-performance liquid chromatography-tandem mass spectrometry in whole blood

Here we describe a liquid chromatography-tandem mass spectrometry method for the blood determination of tricyclic antidepressant drugs (amitriptyline, clomipramine, trimipramine, and imipramine, doxepin, mianserin, maprotyline, dosulepine, amoxapine), their active metabolites (desipramine, nortriptyline, demethylclomipramine, and nordoxepin), and monoamine oxidase inhibitors (toloxatone and moclobemide). A nontricyclic antidepressant (viloxazine) and two other anxiolytic drugs (buspirone and hydroxyzine) that could be encountered in toxicology have been included to this method. After a liquid-liquid extraction from blood, the compounds and their internal standard (methylrisperidone) were eluted on a XTerra RP18 column with a gradient of acetonitrile/ammonium formate buffer 4 mmol/L (pH 3.2). They were then detected by electrospray ionization mass spectrometry with multiple reaction monitoring mode. The calibration curves were linear over the range 5-100 ng/mL. The limit of quantification was 2 ng/mL for each compound. The bias were lower than 10%. Intraday and interday precisions, expressed as variation coefficient, were lower than 13%. The extraction recoveries were between 60 and 80% except for moclobemide, viloxazine, and toloxatone (10-60%). This specific and sensitive method allows management of intoxication and is suitable for the routine determination of antidepressants in forensic investigation.