Kwenga Sichilongo

Comparative chromatography-mass spectrometry studies on the antiretroviral drug nevirapine-Analytical performance characteristics in human plasma determination

A contrast between the analytical performance characteristics using gas chromatography-mass spectrometry (GC-MS) liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-ultraviolet (LC-UV) detection for the determination of the antiretroviral drug (ARV) nevirapine (NVP) in fortified human plasma after QuEChERS extraction has been made. Analytical performance characteristics, i.e. linearities, instrument detection limits (IDLs), limits of quantitation (LOQs), method detection limits (MDLs), % mean recoveries and the corresponding relative standard deviations (%RSDs) were estimated using techniques above. Using GC-MS, the correlation coefficients (r(2)) were ≥0.990, which were deemed acceptable linearities. The MDLs ranged between 11.1-29.8μg/L and 13.7-36.0μg/L using helium and hydrogen carrier gases respectively. The LOQs ranged between 16.5-66.7μg/L and 28.4-98.7μg/L using helium and hydrogen carrier gases respectively with a % mean recovery of 83% and %RSD of 4.6%. Using LC-MS and LC-UV, the correlation coefficients (r(2)) were ≥0.990. The MDLs were ranged between 3.14 and 47.1μg/L. The LOQs ranged between 2.85 and 90.0μg/L respectively. The MDLs using GC-MS, LC-MS and LC-UV were below the therapeutic range for NVP in human plasma is considered to be between 2300μg/L (Cmin) and 8000μg/L (Cmax). This study also demonstrated that helium can be substituted with hydrogen which is relatively cheaper and easily obtainable even by use of a generator.

A sensitive LC-MS/MS method employing a THF–water solvent system for the determination of chloramphenicol, thiamphenicol and florfenicol in bovine muscle

A THF–water solvent system was used in the development of a method for the LC-MS/MS determination of chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FFC) in bovine muscle. The tetrahydrofuran (THF)–water solvent system was recently demonstrated to possess superior figures of merit compared to either the methanol–water or acetonitrile–water solvent systems which are almost exclusively used in LC-MS for the determination of fenicols in food producing animals. The figures of merit included ease of de-solvation when electrospray ionization (ESI) is employed in the negative mode thus leading to more intense mass spectral signals. This phenomenon was shown to be due to greater association of the methyl groups in methanol or acetonitrile with the amide nitrogen common in all the three fenicols. This association is absent in a THF–water solvent system since it is devoid of methyl groups that can easily interact with the amide nitrogen. As a result of the use of the THF–water solvent system, the method detection limits (MDLs) were 0.047, 2.1 and 4.3 μg kg−1 for CAP, TAP and FFC respectively while the limits of quantitation were 0.141, 6.3 and 12.9 μg kg−1 for CAP, TAP and FFC respectively. The decision limits i.e. CCα values according to the Commission Decision 2002/657/EC criteria were 0.36, 50 and 111 μg kg−1 for CAP, TAP and FFC respectively. Linearities were also within acceptable values i.e. 0.9983, 0.9916 and 0.9996 for CAP, TAP and FFC respectively.

A LIQUID CHROMATOGRAPHY MASS SPECTROMETRY METHOD FOR THE DETERMINATION OF SOME PHARMACOLOGICALLY ACTIVE SUBSTANCES VALIDATED USING MILK AS A MATRIX

Two QuEChERS kits originally designed for the extraction of pesticides in foods and foods of plant origin have been compared for the extraction efficiency of different analytes other than pesticides. Six veterinary drugs that have been prohibited for use in foods of animal origin were studied using milk as a matrix and LC-MS as a separation and detection mode. The Association of Official Analytical Chemists (AOAC) and the European Standard EN QuEChERS kits were compared after optimization of key parameters for extraction of chloramphenicol, metronidazole, chlorpromazine, dimetridazole, ronidazole, and dapsone. The EN QuEChERS kit was superior over the AOAC kit in terms of recoveries: (in brackets are % recoveries) chloramphenicol (79), metronidazole (55), chlorpromazine (93), dimetridazole (97), ronidazole (65), and dapsone (69). Using the AOAC kit gave the following values: (in brackets are % recoveries) chloramphenicol (71), metronidazole (66), chlorpromazine (60), dimetridazole (49), ronidazole (37), and dapsone (33). Additionally, three mass spectrometry scan modes, that is, full scan, SIM, and MS/MS were compared. Results showed that SIM was superior over full scan and MS/MS as in the case of chloramphenicol whose method detection limit (MDL) value was 16.3 µg/kg in the SIM mode and 28.2 µg/kg in the full scan mode.

A method employing SPE, MRM LC-MS/MS and a THF–water solvent system for the simultaneous determination of five antiretroviral drugs in human blood plasma

A multiple reaction monitoring liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for the simultaneous determination of five antiretroviral drugs i.e. emtricitabine, tenofovir, efavirenz, lopinavir and ritonavir using commercially sterilized human blood plasma as the matrix and a THF/water solvent system was developed for therapeutic drug monitoring purposes and partially validated using the United States Food and Drug Administration (US FDA) guidelines. The analytical performance characteristics that were examined were limits of detection (LODs), lower limits of quantification (LLOQs), upper limits of quantification (ULOQs), selectivity, percent mean recoveries, precision measured as percent relative standard deviations and accuracy. Optimized LC-MS/MS parameters were employed for this purpose. The percent mean recoveries were between 77.3 and 90.1% using solid phase extraction (SPE) for sample preparation. These recoveries were obtained at three spike levels i.e. the LOD, the LLOQ and the ULOQ. The precision of the SPE technique was within an acceptable range of <15% i.e. between 2.7 and 9.2% at the LLOQ. Accuracy was calculated as the percent deviation of the mean value from the true value and the values ranged between 9.9 and 22.7%. The SPE technique satisfied all the requirements of the US FDA guidelines except for efavirenz whose accuracy was slightly higher than recommended at the LLOQ i.e. 22.7% as opposed to 20%. The limits of detection using SPE also fell below the clinically relevant therapeutic range (3–8 mg L−1) for most of the drugs and therefore the method could be useful for therapeutic drug monitoring in HIV patients.

An Investigation of Liquid Chromatography-Mass Spectral Attributes and Analytical Performance Characteristics of Tenofovir, Emtricitabine and Efavirenz in Human Plasma.

Liquid chromatography (LC) and mass spectral behavior and analytical performance characteristics of efavirenz (EFV), emtricitabine (EMT) and tenofovir (TFV), i.e., individual components of Atripla(®), were probed. This was followed by estimation of their analytical performance characteristics employing LC and a parallel direct infusion sample introduction procedure. Performance characteristics using both types of sample introduction procedures were compared. Using liquid chromatography-mass spectrometry (LC-MS), linearities, i.e., correlation coefficients of the calibration curves of EFV, EMT and TFV, ranged between 0.9300 and 0.9990 in the full scan, selected ion monitoring and mass spectrometry/mass spectrometry (MS-MS) modes. The limits of detection (LODs) ranged between 0.5 and 11.6 µg/L. The lower limits of quantification (LLOQs) and the upper limits of quantification (ULOQs) were in the ranges of 0.9-23.2 and 1.6-38.7 µg/L, respectively. The LODs ranged between 0.8 and 114.7 µg/L. The LLOQs and the ULOQs were in the ranges of 1.6-29.4 and 2.7-49.0 µg/L, respectively. In the case of EMT, sodiated molecular ion at m/z 270 was used to adduce analytical performance characteristics from which lower detection limits were obtained compared with those in the literature where [M+H](+) at m/z 248 was used.

Comparison of New Ultrasonic Digestion Approaches for Plant Matrices in the Analysis of Trace Metals by Inductively Coupled Plasma–Optical Emission Spectroscopy Analysis: Contrast with USEPA Method 3050B

ABSTRACT An ultrasonic method using two approaches, A and B, along with a reference Environmental Protection Agency (EPA) Method 3050B [i.e., a mixture of 30 mL of nitric acid–hydrochloric acid–hydrogen peroxide–water (HNO3-HCl-H2O2-H2O)] were contrasted for leaching of a plant matrix. The trace metals were arsenic, cadmium, cobalt, chromium, copper, mercury, manganese, nickel, lead, selenium, and zinc (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Se and Zn) and quantified by ICP-OES followed by an investigation into residue formation and the impact of digestion time. Approach B was the most accurate and precise with percent recoveries ranging between 99 and 120%, whereas ultrasonic approach A and the USEPA method 3050B gave similar results with poor accuracies and precisions. In the optimization of the digestion time using approach B, the total metal recovery was fairly the same over a period of 120 min except for Cr and Cu, which showed slight variations.

Pre-electrospray ionisation manifold methylation and post-electrospray ionisation manifold cleavage/ion cluster formation observed during electrospray ionisation of chloramphenicol in solutions of methanol and acetonitrile for liquid chromatography-mass spectrometry employing a commercial quadrupole ion trap mass analyser.

We have observed unusual mass spectra of chloramphenicol (CAP) in solutions of methanol or acetonitrile showing intense ions at m/z 297, m/z 311, m/z 325 and m/z 339. The ions observed were different from those which are traditionally observed in the full scan electrospray ionization (ESI) mass spectra of CAP with ions of m/z 321, m/z 323 and m/z 325. We have evidence to show that this process starts with off-line methylation of CAP in solutions of methanol or acetonitrile to give m/z 339. Investigations using nuclear magnetic resonance spectroscopy showed that there is a methylene group somewhere within the CAP molecule but not attached to any of the carbon atoms when the CAP is dissolved in methanol or acetonitrile before infusion into the mass spectrometer. The possible locations of attachment were speculated to be the electronegative atoms apart from the chlorine atoms due to valence considerations. The methylene group is attached to the nitrogen atom and forms a bond as observed in the MS/MS spectra of m/z 297, m/z 311, m/z 325 and m/z 339 which give m/z 183 as the base peak in all cases. Further experiments showed that there is cleavage of the methylated CAP molecule followed by cluster ion formation involving addition of methylene groups to the CAP fragment with m/z 183 to produce ions of m/z including 297, 311, 325 and 339. This process occurs in the mass spectrometer in the region housing the tube lens and is triggered when the ions are accelerated through this region by application of a negative tube lens offset voltage. This region affords collision of the charged droplets with a collision gas in this case nitrogen to strip the droplets of their solvent molecules. Experiments to follow the intensities of m/z 183, m/z 311, m/z 321, m/z 323, m/z 325 and m/z 339 as the tube lens offset voltage was varied were done in which the intensities of m/z 311, m/z 325 and m/z 339 were observed to be at their peak when the tube lens offset voltage was set at −40 V. When the tube lens offset voltage is swung to +40V, thus decelerating the ions through the capillary skimmer region via the tube lens, the traditionally observed spectra with m/z 321, m/z 323 and m/z 325 were observed.

Comparison of efficiencies of selected sample extraction techniques for the analysis of selected antiretroviral drugs in human plasma using LC-MS

INTRODUCTION Sample preparation in bio analytical chemistry poses a challenge because it can be compound dependent. We compared six sample extraction techniques i.e. QuEChERS (Q), liquid extraction (LE), protein precipitation (PPT), Q-PPT, Q-LE and LE-PPT for the extraction of antiretroviral drugs emtricitabine, tenofovir, efavirenz, lopinavir and rotinavir in human blood plasma. METHOD A multiple reaction monitoring liquid chromatography- tandem mass spectrometry method for the determination of the same antiretroviral drugs developed and validated in this laboratory was used. Comparisons were based on the efficiencies of extraction, the precisions and accuracies. Using United States Food and Drug Administration guidelines, analytical performance characteristics i.e. limits of detection, lower limits of quantification and upper limits of quantification were also compared. RESULTS The percent mean recoveries ranged between 68.8 and 81.2% for single modes and 52.4-70.5% for mixed mode techniques. The precisions of all the extraction techniques were within the Using United States Food and Drug Administration guidelines acceptable range of <15% at all concentration levels for all analytes. Accuracy ranged between 8.73 and 65.94% for single mode techniques and between 21.73 and 51.59% for mixed mode techniques. DISCUSSION The mixed modes gave slightly lower recoveries but Q-LE compared well with the single modes at slightly higher spike levels. Limits of detection for all the six sample preparation techniques fell below the clinically relevant therapeutic range of approximately 3-8ppm. Therefore all techniques can be employed for routine therapeutic drug monitoring studies.