Chinmoy Ghosh

Influence of ionization source design on matrix effects during LC-ESI-MS/MS analysis.

Glycerophosphocholines (GPChos) are known to cause matrix ionization effects during the analysis of biological samples (i.e. plasma, urine, etc.) in LC-MS/MS. In general, such matrix effect is directly related to an insufficient sample clean-up of the biofluids. In addition to GPCho; design of ionization source and/or LC also plays a very important role in matrix effects. In this research paper, different types of matrix effects, i.e. ion suppression or enhancement were observed in differently designed ion sources coupled with different LCs, from the same molecule, acamprosate (ACM), under the same chromatographic conditions. ACM was analyzed in a negative polarity in electrospray ionization interface using Z-spray and orthogonal spray ion source design. The analyte showed almost complete ion suppression in the Z-spray ionization source coupled with UPLC/HPLC, whereas there was very little ion enhancement in the orthogonal spray ionization source coupled with HPLC. In both the cases different GPChos were responsible, as evident from the presence of m/z 815.4 in Z-spray ion source and m/z 759.0 in orthogonal spray ion source. Hence, this approach can be used to evaluate the matrix effects in plasma samples during development and validation of LC-MS/MS method of drugs and their metabolites in different biological matrixes.

Preservation of Bioavailability of Ingredients and Lack of Drug-Drug Interactions in a Novel Five-Ingredient Polypill (Polycap™)

BackgroundThe Polycap™ (polypill; aspirin [acetylsalicylic acid], ramipril, simvastatin, atenolol, and hydrochlorothiazide) was found to be safe and effective for reducing multiple cardiovascular risk factors in The Indian Polycap™ Study (TIPS).ObjectiveWe evaluated the bioavailability of each ingredient of the Polycap™ and determined any drug-drug interactions relative to single component reference preparations.MethodsThe bioavailability of the ingredients of the Polycap™ (T; test) when formulated as a single capsule was compared with that of identical capsules with each of its ingredients administered separately (R; reference) in a five-arm, randomized, single-dose, two-period, two-treatment, two-sequence, crossover trial with at least a 2-week washout period in a total of 195 healthy volunteers. Plasma concentrations of each drug and, where applicable, its active metabolite were measured using validated liquid chromatographytandem mass spectrometry and ultra-performance liquid chromatography. Mean pharmacokinetic parameters and their standard deviations were computed for each analyte.ResultsComparative bioavailability was computed and no drug-drug interactions and no difference in comparative bioavailability were concluded for each ingredient based on point estimates of the T/R ratio of the geometric means falling within 80–125% for peak plasma concentration (Cmax), area under the plasma concentration-time curve from time zero to the last measurable concentration (AUCt), and AUC from time zero to infinity (AUC∞). The T/R ratio for Cmax, AUCt and AUC∞ was within 80–125% for atenolol, hydrochlorothiazide, ramipril, ramiprilat and dose-normalized salicylic acid. However, for simvastatin, the T/R point estimates for Cmax, AUCt and AUC∞ for Ln-transformed data were significantly lower (∼3–4%) than the lower bound of 80%. For its active metabolite, simvastatin acid, these estimates were significantly higher (∼25–35%) than the higher bound of 125%. Thus, the increased bioavailability of active simvastatin acid appeared to compensate for the loss of bioavailability of simvastatin.ConclusionThe Polycap™ was found to be effective and safe in the previously published TIPS trial. The present study in healthy volunteers establishes that Polycap™ is safe (no serious adverse events) and well tolerated, and that there is no indication of pharmacokinetic drug-drug interactions for any of the ingredients, with their bioavailabilities being well preserved.

Determination of pregabalin in human plasma by electrospray ionisation tandem mass spectroscopy

A rapid, precise, specific, and accurate Electrospray Ionisation Tandem Mass Spectrometry (ESI-MS / MS) method has been developed and subsequently validated, for the determination of pregabalin (PB) in human plasma. Gabapentin (GB) was used as the internal standard. PB and GB were extracted from the plasma using a combination of deproteinization, using 0.1% formic acid and liquid–liquid extraction, using methylene chloride. PB and GB were separated using the Hypurity advance column (50 mm × 4.6 mm, 5 μm) and mobile phase, consisting of methanol : 0.1% formic acid (80:20 v / v). PB was determined by using ESI-MS / MS in positive ion mode, with the help of the API 2000 spectrophotometer, operated in a multiple reaction monitoring mode. The parent-to-product ion combination of m / z 160.2→55.1 and 172.2→95.0 was used to quantify PB and GB, respectively. The assay was validated in the concentration range of 99.79 – 4019.90 ng / mL for PB. The limit of quantification (LOQ) was identifiable and reproducible at 99.79 ng / mL. The method has been successfully applied to study the pharmacokinetics of PB in healthy male volunteers.

Simultaneous estimation of atorvastatin and its two metabolites from human plasma by ESI-LC-MS/MS

A selective, sensitive, and fast high performance liquid chromatography (HPLC) method with mass spectrometric (MS) detection mode has been developed and validated completely in human plasma. Atorvastatin (ATO), p-hydroxy atorvastatin (p-HATO), o-hydroxy atorvastatin (o-HATO) and internal standard (IS) are extracted from human plasma via solid phase extraction (SPE) technique. After elution, the solution is evaporated, then reconstituted with 250 µL of Mobile Phase and analyzed using HPLC/MS/MS system. An isocratic mode is used to separate interference peaks using a Symmetry C-18, 75 × 4.6 mm ID, 3.5 µ, column. The m/z of ATO, o-HATO and p-HATO are 559.2/440.2, 575.3/440.4 and 575.0/440.4 respectively. Linearity ranges are 0.05 to 252.92 ng/mL for ATO, p-HATO and o-HATO respectively. Calibration functions, lower limit of quantitation (LLOQ), stability, intra- and inter-day reproducibility, accuracy, and recovery are estimated. This method is free from matrix effects and any abnormal ionization. This method was successfully applied to a single dose 80 mg tablet bioequivalence (BE) study of Atorvastatin. Copyright

Ionization Polarity as a Cause of Matrix Effects, its Removal and Estimation in ESI-LC-MS/MS Bio-analysis

Matrix effect is the effect on an analytical method caused by all other components of the sample except the specific compound to be quantified. Matrix effects and selectivity issues have long been associated with bioanalytical techniques. A number of approaches have been investigated to improve reproducibility and robustness of LC-MS-MS methods that are subjected to matrix effects. In the present research work the role of ionization polarity on matrix effect was studied. Enalapril and its metabolite were analyzed in positive and negative polarity by using ESI-LC-MS/MS. Matrix factor (MF) was determined to evaluate the matrix effects in different polarities. Two different concentration levels of each analyte were used to determine the MF. In positive polarity the MF at two different concentration levels were 0.6353 & 0.6496 for enalapril and 0.6885 & 0.6770 for enalaprilat, whereas, the MF in negative polarity at two different concentration levels were 0.8203 & 0.7717 for enalapril and 1.1124 & 1.0915 for enalaprilat. These data showed approximately 30-35% ion suppression in positive polarity for both the analyte, but approximately 20% ion suppression for enalapril and 10% ion enhancement for enalaprilat in negative polarity. So, matrix effects depend on the ionization polarity also.

A LC-MS analysis of acamprosate from human plasma: pharmacokinetic application

A rapid and highly sensitive method for the determination of acamprosate (ACM), in human plasma using ESI-LC-MS/MS (electrospray ionization liquid chromatography tandem mass spectrometry) in negative ionization polarity in multiple reactions monitoring (MRM) mode was developed and validated. The procedure involves a simple protein precipitation step. Chromatographic separation was carried out on a Hypersil BDS C(18) column (150 mm × 4.6 mm, 5 µm) with an isocratic mobile phase and a total run time of 2.5 min. The standard calibration curves were linear within the range of 7.04-702.20 ng/mL for ACM (r ≥ 0.990). This study briefly describes the role of ion source design on matrix effects. ACM shows matrix effects in z-spray ionization source design, whereas it has no matrix effects in orthogonal spray ion source design. This method was successfully applied to a pharmacokinetic study after oral administration of acamprosate 333 mg tablet in Indian healthy male volunteers.

A rapid and most sensitive liquid chromatography/tandem mass spectrometry method for simultaneous determination of alverine and its major metabolite, para hydroxy alverine, in human plasma: application to a pharmacokinetic and bioequivalence study

A rapid and highly sensitive method for the determination of alverine (ALV) and its metabolite, para hydroxy alverine (PHA), in human plasma using LC-MS/MS in positive ion electrospray ionization (ESI) in multiple reactions monitoring (MRM) mode was developed and validated. The procedure involves a simple solid phase extraction (SPE). Chromatographic separation was carried out on a Hypersil GOLD C(18) column (50 mm x 4.6 mm, 5 microm) with an isocratic mobile phase and a total run time of 1.5 min. The standard calibration curves showed excellent linearity within the range of 0.060-10.051 ng/mL for ALV and 0.059-10.017 ng/mL for PHA (r > or = 0.990). This method was successfully applied to a pharmacokinetic study after oral administration of alverine citrate 120 mg capsule in Indian healthy male volunteers.

Rapid and sensitive liquid chromatography/tandem mass spectrometry method for simultaneous determination of enalapril and its major metabolite enalaprilat, in human plasma: application to a bioequivalence study.

A rapid and most sensitive method for simultaneous determination of enalapril (ENP) and its metabolite, enalaprilat (ENPT), in human plasma using ESI-LC-MS/MS (electrospray ionization liquid chromatography tandem mass spectrometry) positive ion multiple reactions monitoring (MRM) mode, was developed and validated. The procedure involves a simple solid-phase extraction (SPE) followed by evaporation of the sample. Chromatographic separation was carried out on a Hypurity C(18) column (50 mm × 4.6 mm, 5 µm) with an isocratic mobile phase and a total run time of 2.0 min only. The MRM of ENP and ENPT is 377.10 → 234.20 and 349.20 → 206.10 respectively. The standard calibration curves showed excellent linearity within the range of 0.064 to 431.806 ng/mL for ENA and 0.064 to 431.720 ng/mL for ENPT (r ≥ 0.990). This is the only method which can quantitate upto 0.064 ng/mL for both ENP and ENPT in a single run with the shortest analysis time. In matrix effect experiment, this method shows a % CV (% coefficients of variation) of less than 5, which means that the proposed method is free from any kind of irregular ionization process. This method was successfully applied to a pharmacokinetic study after oral administration of enalapril maleate 20 mg tablet in Indian healthy male volunteers.

SIMULTANEOUS DETERMINATION OF ASPIRIN AND ITS METABOLITE FROM HUMAN PLASMA BY UPLC-UV DETECTION: APPLICATION TO PHARMACOKINETIC STUDY

A rapid and sensitive liquid chromatography method with UV detection for simultaneous measurement of aspirin (ASA) and salicylic acid (SA) was developed and validated completely in human plasma. ASA, SA, and Benzoic acid (BA) as internal standard were extracted via protein precipitation with Perchloric acid. An isocratic elution with binary mode was used to separate interference peaks using a C18 Acquity column with only three minutes of analysis time. The linearity range was 15 to 6000 n g mL−1. Calibration functions, LOQ, stability, intra- and inter-day reproducibility, and accuracy were estimated. The inter- and intra-day % CV for ASA and SA are ±15% and the percentage change of all stability samples with comparison samples were within 10%. The in vitro conversion of ASA to SA was also studied and it was controlled to keep at a minimum (<10%). With respect to other published methods this method is most sensitive in UV detection, as well as its sensitivity and throughput is comparable or even better than published LC-MS/MS methods. This method was successfully applied to a single dose Bioequivalence (BE) study of following administration of 81 mg enteric coated Aspirin tablets.

SIMULTANEOUS DETERMINATION OF DOCOSAHEXAENOIC ACID AND EICOSAPENTAENOIC ACID BY LC-ESI-MS/MS FROM HUMAN PLASMA

A sensitive and rapid method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) with simple single step protein precipitation has been developed and validated for the quantitative determination of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human plasma. After addition of internal standard to human plasma, samples were extracted by simple protein precipitation using acetonitrile as the precipitating agent. The extracts were analyzed by HPLC with the detection of the analyte in the multiple reaction monitoring (MRM) mode. This method for the simultaneous determination of DHA and EPA is accurate and reproducible, with limits of quantitation of 50.00 ng/mL in plasma. The standard calibration curves for both DHA and EPA are linear (r > 0.99) over the concentration ranges 50.00–7498.50 ng/mL in human plasma, respectively. The intra- and inter-day precision over the concentration range for DHA and EPA are less than 10.16 and 6.72 (relative standard deviation, %RSD), and accuracy is between 91.17–104.74% and 95.81–108.33%, respectively.