Very complex internal standard response variation in LC-MS/MS bioanalysis: root cause analysis and impact assessment.

Internal standards (ISs) are essential for the development and use of reliable quantitative bioanalytical LC-MS/MS methods, because they correct for fluctuations in the analytical response that are caused by variations in experimental conditions. Sample-to-sample differences in the IS response are thus to be expected, but a large variability often is an indication of nonoptimal sample handling or analysis settings. This paper discusses a number of cases of very complex variation of IS responses that could be attributed to analytical problems such as injection errors and sample inhomogeneity, and matrix-related issues such as degradation and increased ionization efficiency. A decision tree is proposed to help find the underlying root cause for extreme IS variability.

Box-Behnken design directed optimization for sensitivity assessment of anti-platelet drugs.

Optimization of electrospray ionization (ESI) parameters is routinely carried out by one factor at a time (OFAT) or auto-tune software (ATS). Design of experiments (DOE) approach has been reported to be an excellent alternative to OFAT or ATS. Box-Behnken Design (BBD) was successfully used to optimize ESI parameters like nebulizing gas flow rate, desolvation line temperature, heat block temperature, and drying gas flow rate for [M + H]+ intensity of Clopidogrel bisulfate (CLP) and Ticlopidine (TLP). BBD model was found to be significant with p < .0001 for both CLP and TLP. The predicted and optimized (OL) ESI parameters were used for chromatographic analysis and were compared against three levels of ESI parameters, i.e. low level (LL), medium level (ML), and high level (HL). The OL ESI parameters were subjected to chromatographic analysis and its mean peak area was significantly higher than mean peak area for LL, ML, and HL ESI in case of CLP and TLP (p < .001). However, no significant difference was observed between the mean peak area for ML and OL of TLP. Thus, BBD can be considered with 29 trials to optimize four mass spectrometric parameters. The liquid chromatographic parameters percentage of methanol, percentage of formic acid and flow rate were also optimized using BBD. However, the optimized method did not significantly influence the peak response over the non-optimized method.

A Review of Analytical Methods for the determination of Clopidogrel in Pharmaceuticals and Biological Matrices.

P2Y12 belongs to a group of G protein-coupled (GPCR) purinergic receptors and is a receptor for adenosine diphosphate (ADP). The P2Y12 receptor is involved in platelet aggregation and acts as a biological target for treating thromboembolisms and other clotting disorders. The use of Clopidogrel (CLO) has improved the morbidity and mortality endpoints including cardiovascular death, recurrent myocardial infarction (MI) and stroke at 30 days after percutaneous coronary intervention (PCI). CLO is one such drug that specifically and irreversibly inhibits the P2Y12 subtype of ADP receptor. This review delivers a detail description of various analytical methods published for the estimation of CLO and its combinations in pharmaceuticals and biological matrices. The review highlights the basic as well as advanced techniques performed for estimating CLO. The most commonly used assay techniques were UV and Visible spectrophotometry, high performance liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC), micellar liquid chromatography (MLC), micellar electro kinetic chromatography (MEKC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Despite other analytical methods employed for the assay of CLO, the review reveals that the technique of HPLC with UV detection was widely used.

Development of a high-performance liquid chromatography method for the simultaneous determination of chiral impurities and assay of (S)-clopidogrel using a cellulose-based chiral stationary phase in methanol/water mode.

A simple reversed-phase high-performance liquid chromatography method for the chiral separation of the active pharmaceutical ingredient (S)-clopidogrel has been developed on the cellulose-based Chiralcel OJ-RH chiral stationary phase. The S enantiomer was baseline resolved from its R impurity (impurity C) with a mobile phase consisting of methanol/water (100:15) without any interference coming from the other two potential chiral impurities A and B. The enantio- and chemoselective method was partially validated and compared with that reported in the United States Pharmacopoeia for the drug product. The versatility of the Chiralcel OJ-RH allowed separating the enantiomers of the impurity B also under normal phase and setting up an efficient strategy to convert the racemic sample into the enantiomeric S form on a semipreparative scale.

Raman chemical imaging for spectroscopic screening and direct quantification of falsified drugs.

Falsified drugs are a threat to the health of patients. The analytical control of such products contributes to the fight against this global issue. Raman chemical imaging is a method that relies on consecutive measurements at the surface of a sample, combining spectroscopy, microscopy and chemometrics. This article explores the capabilities of this analytical technique proposing an innovative methodology with spectroscopic screening for the identification of chemical compounds and the direct quantification of the active substance (without prior calibration). Two chemometric methods were used: Multivariate Curve Analysis - Alternate Least Squares for the qualitative analysis and Direct Classical Least Squares for the quantitative analysis. The methodology was optimized with samples prepared in the laboratory and validation parameters were studied. The methodology was then applied to real (authentic and falsified) samples of Viagra® and Plavix®. Despite the presence of fluorescence emission in some samples, the methodology succeeded in the detection of active pharmaceutical ingredients, and in the discrimination of three salts of clopidogrel (in generic formulations of Plavix®). The quantitative deviation from the reference method ranged from -15% to +24% of the active substance content. This deviation may be considered to be acceptable since it is sufficient for assessing the risk to the health of patients and for quickly alerting the health authorities.

New approach application of data transformation in mean centering of ratio spectra method.

Most of mean centering (MCR) methods are designed to be used with data sets whose values have a normal or nearly normal distribution. The errors associated with the values are also assumed to be independent and random. If the data are skewed, the results obtained may be doubtful. Most of the time, it was assumed a normal distribution and if a confidence interval includes a negative value, it was cut off at zero. However, it is possible to transform the data so that at least an approximately normal distribution is attained. Taking the logarithm of each data point is one transformation frequently used. As a result, the geometric mean is deliberated a better measure of central tendency than the arithmetic mean. The developed MCR method using the geometric mean has been successfully applied to the analysis of a ternary mixture of aspirin (ASP), atorvastatin (ATOR) and clopidogrel (CLOP) as a model. The results obtained were statistically compared with reported HPLC method.

Comparative study of three modified numerical spectrophotometric methods: an application on pharmaceutical ternary mixture of aspirin, atorvastatin and clopedogrel.

Three novel numerical methods were developed for the spectrophotometric multi-component analysis of capsules and synthetic mixtures of aspirin, atorvastatin and clopedogrel without any chemical separation. The subtraction method is based on the relationship between the difference in absorbance at four wavelengths and corresponding concentration of analyte. In this method, the linear determination ranges were 0.8-40 µg mL(-1) aspirin, 0.8-30 µg mL(-1) atorvastatin and 0.5-30 µg mL(-1) clopedogrel. In the quotient method, 0.8-40 µg mL(-1) aspirin, 0.8-30 µg mL(-1) atorvastatin and 1.0-30 µg mL(-1) clopedogrel were determine from spectral data at the wavelength pairs that show the same ratio of absorbance for other two species. Standard addition method was used for resolving ternary mixture of 1.0-40 µg mL(-1) aspirin, 0.8-30 µg mL(-1) atorvastatin and 2.0-30 µg mL(-1) clopedogrel. The proposed methods were validated. The reproducibility and repeatability were found satisfactory which evidence was by low values of relative standard deviation (<2%). Recovery was found to be in the range (99.6-100.8%). By adopting these methods, the time taken for analysis was reduced as these methods involve very limited steps. The developed methods were applied for simultaneous analysis of aspirin, atorvastatin and clopedogrel in capsule dosage forms and results were in good concordance with alternative liquid chromatography.

HPLC-PDA-ORD bioassay of S-(+) and R-(-) clopidogrel on rat dried blood spots.

A simple and rapid chiral high-performance liquid chromatography (HPLC) method was developed and validated for bioanalysis of clopidogrel enantiomers on rat dried blood spots (DBS). Clopidogrel enantiomers were extracted from DBS using ethanol: methanol (80:20, v/v) and separated on a Chiralcel OJ-H column containing cellulose tris (4-methly benzoate) as a polysaccharide stationary phase using n-hexane-ethanol-diethylamine (70:30, 0.1 v/v) as a mobile phase at a flow rate of 1.0 mL/min. The detection was carried out at 220 nm using a photodiode array (PDA) detector while the elution order of the enantiomers was determined by a polarimeter connected to PDA in series. The effect of hematocrit on extraction of clopidogrel enantiomers from DBS was evaluated and no interference from endogenous substances was noticed. The overall accuracy of (R) and (S) enantiomers of clopidogrel from DBS were 91.6 and 89.2%, respectively. The calibration curves were linear over the concentration range of 1-500 µg/mL for both enantiomers. The results show that the method is specific, precise, and reproducible (intra- and interday precision relative standard deviations (RSDs) <10.0%). The stability of racemic clopidogrel was performed under all storage conditions and the results were found to be well within the acceptance limits.

[Determination of clopidogrel sulfate in pharmaceutical formulation and biological fluids by extraction spectrofluorimetric method].

A simple and sensitive spectrofluorimetric method has been developed for the determination of clopidogrel sulfate in pharmaceutical formulation and human urine/serum. It is based on the formation of ion-pair complex between clopidogrel sulfate and alizarin red in 0.3 mol x L(-1) hydrochloric acid solution. The ion pair complex was extracted in dichloromethane and the fluorescence intensity was measured at 550 nm after excitation at 428 nm. The various factors influencing ion-pair complex formation and fluorescence determination were discussed. Under the optimized conditions, the fluorescence value showed a good linear relationship with the clopidogrel sulfate concentration ranging from 1.0 to 11.0 µg x mL(-1). The equation of calibration curve was F = 53.32 + 35.01c (µg x mL(-1)), r = 0.994, and the detection limit was found to be 0.11 µg x mL(-1). No interference was observed from common co-existing substances or pharmaceutical excipient. The determination recoveries of clopidogrel sulfate in pharmaceutical formulation and human urine/serum samples were 90.6%-99.3%, 104.6%-109.3%, 96.3%-105.0%, respectively. The method was successfully applied to detect clopidogrel sulfate in clopidogrel sulfate tablet and human urine/ serum. The obtained results of clopidogrel sulfate tablet were in good agreement with the results of HPLC. Therefore, it is concluded that the proposed method is simple, sensitive and rapid for the determination of clopidogrel sulfate in real samples.

Performance assessment of microflow LC combined with high-resolution MS in bioanalysis.

BACKGROUND: There continues to be consistent pressure for bioanalytical scientists to achieve lower limits of quantitation. The reasons range from smaller sample volumes available for analysis, to more potent analytes and the growth of biologics in drug development. This has led scientists to investigate alternative LC techniques, including microflow and nanoflow. These techniques have been shown to increase sensitivity of electrospray methods and reduce ionization matrix effects. Because high-resolution MS has significant benefits for the analysis of biologics, this type of mass spectrometer is becoming increasingly important in bioanalysis. RESULTS: For microflow analysis, a new ion source and significant extra sample preparation or chromatographic separation are not required. However, increased sensitivity and reduced matrix effects were consistently demonstrated when compared with UHPLC flow rates. The extent of matrix effects observed were compound dependent. DISCUSSION: This paper presents the utility of combining high-resolution/accurate mass with microflow LC from a quantitative standpoint. This includes evaluating the typical quantitative parameters of sensitivity, linearity/dynamic range, precision and accuracy. It also includes the evaluation of changes in signal suppression using microflow LC and microspray ionization. The benefits and disadvantages of using the combination of these two technologies for quantitative bioanalysis are also discussed.

Detection and characterization of ticlopidine conjugates in rat bile using high-resolution mass spectrometry: applications of various data acquisition and processing tools.

Ticlopidine, an antiplatelet drug, undergoes extensive oxidative metabolism to form S-oxide, N-oxide, hydroxylated and dealkylated metabolites. However, metabolism of ticlopidine via conjugation has not been thoroughly investigated. In this study, multiple data acquisition and processing tools were applied to the detection and characterization of ticlopidine conjugates in rat bile. Accurate full-scan mass spectrometry (MS) and collision-induced dissociation (CID) MS/MS data sets were recorded using isotope pattern-dependent acquisition on an LTQ/Orbitrap system. In addition, mass spectral data from online H/D exchanging and high collision energy dissociation (HCD) were recorded. Data processes were carried out using extracted ion chromatography (EIC), mass defect filter (MDF) and isotope pattern filter (IPF). The total ion chromatogram displayed a few major conjugated metabolites and many endogenous components. Profiles from EIC and IPF processes exhibited multiple conjugates with no or minimal false positives. However, ticlopidine conjugates that were not predictable or lost a chorine atom were not found by EIC or IPF, respectively. MDF was able to detect almost all of ticlopidine conjugates although it led to a few more false positives. In addition to CID spectra, data from HCD, H/D exchanging experiments and isotope pattern simulation facilitated structural characterization of unknown conjugates. Consequently, 20 significant ticlopidine conjugates, including glucuronide, glutathione, cysteinylglycine, cysteine and N-acetylcysteine conjugates, were identified in rat bile, a majority of which are associated with bioactivation and not previously reported. This study demonstrates the utility and limitation of various high-resolution MS-based data acquisition and processing techniques in detection and characterization of conjugated metabolites.

Quantitative determination of clopidogrel and its metabolites in biological samples: a mini-review.

Clopidogrel has been applied in antiplatelet therapy since 1998 and is the thienopyridine with the largest clinical experience. By 2011, clopidogrel (Plavix(®)) was the second top-selling drug in the world. Following complete patent expiry in 2012/2013 its use is expected to grow even further from generics entering the market. Prefaced by a brief description of clopidogrel metabolism, this review analyzes analytical methods addressing the quantification of clopidogrel and its metabolites in biological samples. Techniques that have been applied to analyze human plasma or serum are predominantly LC-MS and LC-MS/MS. The lowest level of clopidogrel quantification that has been achieved is 5pg/mL, the shortest runtime is 1.5min and almost 100% recovery has been reported using solid-phase extraction for sample preparation.

In vitro biotransformation studies of 2-oxo-clopidogrel: multiple thiolactone ring-opening pathways further attenuate prodrug activation.

The biotransformation of clopidogrel has been under extensive investigation to address the observed high clinical variability and resistance of its antithrombotic prodrug therapy. Clopidogrel (M0) is first activated to its thiolactone intermediate, 2-oxo-clopidogrel (M2), by hepatic cytochrome P450 (P450) enzymes. Subsequent P450-catalyzed S-oxidation is followed by thioester hydrolysis, which cleaves the thiolactone ring and yields a sulfenic acid intermediate (M12); this intermediate is reduced to the final active metabolite (M13). The aim of the present study is to characterize the metabolic fates of M2 more comprehensively with focus on the thiolactone ring-opening pathways. It was found that the bioactivating S-oxidation confers on the thiolactone moiety not only one electrophilic site at the carbonyl C-atom (Site A), but also a second one at the allylic bridge C-atom (Site B). Both sites can react with H2O or other nucleophiles, like glutathione (GSH), leading to different thiolactone ring-opening pathways. In addition to the pharmacologically desired A-H2O pathway leading to M13 formation, the A-GSH pathway leads to the formation of a glutathione conjugate (GS-3), the B-H2O pathway leads to the formation of a desulfurized hydroxyl metabolite (M17), and the B-GSH pathway leads to the formation of a desulfurized glutathione conjugate (GS-2). These results demonstrate the reactive nature of the electrophilic thiolactone S-oxide intermediate (M11) and suggest that M13 formation from M2 might be accompanied by more attenuating pathways than previously reported. The research presented here may facilitate future studies exploring the clinical antithrombotic response to clopidogrel as well as the susceptibility to the adverse effect of clopidogrel and its close prodrug analogues.

Development and validation of a liquid chromatographic method for purity control of clopidogrel-acetylsalicylic acid in combined oral dosage forms.

A reversed phase liquid chromatographic method with UV detection for the simultaneous determination of clopidogrel and acetylsalicylic acid and their related substances in combined oral formulations was developed and validated. Good separation was achieved on a Luna C18 column (150 mm × 4.6 mm, 3 µm) using gradient elution at a flow rate of 1 mL/min and a column temperature of 35 °C. UV detection was performed at 220 nm. The validation was performed according to the ICH guidelines. The method proved to be specific, sensitive (LOQ=0.975 µg/mL and 0.0384 µg/mL for clopidogrel and acetylsalicylic acid, respectively), linear in the concentration range from LOQ to 325 µg/mL for clopidogrel and from LOQ to 650 µg/mL for acetylsalicylic acid, precise (RSD values for intermediate precision <1%) and accurate with mean recovery values of 100.7% and 100.2% for clopidogrel and acetylsalicylic acid, respectively. Moreover, the solution stability and method robustness were examined. The method gives satisfactory separation of impurities of clopidogrel and acetylsalicylic acid and so it is suitable for quantification of the related substances as well as for the assay of the actives.

Incurred sample reanalysis (ISR): a decisive tool in bioanalytical research.

The AAPS Workshop 2008 on Current Topics in GLP Bioanalysis: Assay Reproducibility for Incurred Samples was the defining moment in establishing incurred sample reanalysis (ISR) as a mandatory exercise in demonstrating assay reproducibility using incurred (study) samples. The importance of ISR can be envisaged from its role in clinical as well as non-clinical studies. Incurred samples can differ significantly in their composition when compared with the calibration standards and quality control samples that are used to validate the developed method. The present article attempts to summarize five troubleshooting cases encountered in the analyses of incurred samples for bioanalytical methods developed in our laboratory for mesalamine, hydrochlorothiazide, clopidogrel, sildenafil and rabeprazole. The issues identified were related to: sample inhomogeneity, sample processing error, impact of buffer pH during sample preparation, instability of metabolite and change in laboratory environment. The steps taken to trace and correct these incidents are discussed with adequate data. These examples will further broaden the scope and emphasize the significance of ISR. We believe this investigation will help to develop more reliable and efficient bioanalytical methods.

An improved method for specific and quantitative determination of the clopidogrel active metabolite isomers in human plasma.

Pharmacokinetic analyses of clopidogrel are hampered by the existence of multiple active metabolite isomers (H1 to H4) and their instability in blood. We sought to retest the pharmacodynamic activities of the four individual active metabolite isomers in vitro, with the ultimate aim of determining the isomers responsible for clopidogrel activity in vivo. In vitro activity was evaluated by measuring binding of [³³P]-2-methylthio-ADP on P2Y12-expressing Chinese hamster ovary (CHO) cells and human platelets in platelet-rich plasma (PRP). A stereoselective method that used reverse-phase ultra high-performance liquid chromatography (UHPLC) and tandem mass spectrometry (MS) was developed to measure individual concentrations of the stable 3'-methoxyacetophenone (MP) derivatives of H1-H4. The new method was used to analyze plasma samples from clopidogrel-treated subjects enrolled in a phase I clinical trial. In vitro binding assays confirmed the previously observed biological activity of H4 (IC50: CHO-P2Y12: 0.12 µM; PRP: 0.97 µM) and inactivity of H3, and demonstrated that H1 was also inactive. Furthermore, H2 demonstrated approximately half of the biological activity in vitro compared with H4. Optimisation of UHPLC conditions and MS collision parameters allowed the resolution and detection of the four derivatised active metabolite isomers (MP-H1 to MP-H4). The stereoselective assay was extensively validated, and was accurate and precise over the concentration range 0.5-250 ng/ml. Only MP-H3 and MP-H4 were quantifiable in incurred clinical samples. Based on in vitro pharmacodynamic data and found concentrations, the active metabolite isomer H4 is the only diastereoisomer of clinical relevance for documenting the pharmacokinetic profile of the active metabolite of clopidogrel.

Simultaneous determination of clopidogrel and aspirin by RP-HPLC from bulk material and dosage formulations using multivariate calibration technique.

A rapid, simple, and easy method for the simultaneous determination of clopidogrel and aspirin from bulk material and dosage formulations in the presence of meloxicam as internal standard has been developed. Separation was carried out on a Purospher star C(18) (5 µm, 250 × 4.6 mm) column at ambient temperature. The mobile phase consisted of methanol-water (80:20, v/v), the pH of the mobile phase was adjusted to 3.4 with ortho-phosphoric acid and pumped at a flow rate of 1 mL/min using isocratic pump system. Multivariate chromatographic calibration technique was subjected to high-performance liquid chromatography (HPLC) data for simultaneous quantitative analysis of binary mixtures of clopidogrel and aspirin. HPLC data based on the analyte peak areas were obtained at five wavelengths (225, 230, 235, 240, and 245 nm). The mathematical algorithm of multivariate chromatographic calibration technique is based on the use of the linear regression equations. Calibration plots for clopidogrel and aspirin were constructed at each wavelength by using the peak areas corresponding to the concentrations of each active compound. This multivariate chromatographic method was also applied to a commercial pharmaceutical dosage form containing clopidogrel and aspirin.

Light transmittance aggregometry induced by different concentrations of adenosine diphosphate to monitor clopidogrel therapy: a methodological study.

Light transmission aggregation (LTA) is considered the reference method to identify residual platelet reactivity (RPR) in high-risk patients with coronary artery disease on clopidogrel treatment. An international standardization of this technique is still ongoing and different concentrations of adenosine diphosphate (ADP) as the agonist for LTA have been used to evaluate the inhibitory effect of clopidogrel treatment. To evaluate RPR, LTA was performed using different ADP concentrations (2, 5, 10, and 20 µmol/L) in 466 high-risk patients with coronary artery disease on dual antiplatelet therapy who underwent percutaneous coronary intervention and in 46 healthy subjects. A VerifyNow P2Y12 assay was assessed as a point-of care system. Imprecision studies showed higher coefficients of variation for LTA by 2 and 5 µmol/L ADP (healthy subjects: 4.7% and 3.9%; patients: 6.8% and 5.2%, respectively) in comparison with those obtained determining LTA using 10 and 20 µmol/L ADP (healthy subjects: 2.2% and 2.3%; patients: 2.7% and 3.1%, respectively). In patients, a significant difference (P < 0.0001) between mean values of LTA obtained with all ADP concentrations was found, even if LTA data were significantly correlated (at least: rho = 0.88, P < 0.0001). However, data from 10 and 20 µmol/L ADP LTA were very similar and highly concordant (k = 95.9%). All agreements were significant (for all P < 0.0001), in particular the agreement between 10 and 20 µmol/L ADP LTA was very good (k = 0.86, P < 0.0001). A moderate agreement between VerifyNow and both 10 and 20 µmol/L ADP LTA was observed. LTA by 10 and 20 µmol/L ADP gave equivalent percentages of aggregation and highly concordant results in terms of RPR in patients with coronary artery disease on clopidogrel. Significant concordant results were observed between both 10 and 20 µM ADP LTA and VerifyNow. This suggests that a concentration of 10 µmol/L ADP may be considered adequate for the identification of RPR of patients on clopidogrel and should be preferred for standardization LTA.

Simultaneous determination of clopidogrel and its carboxylic acid metabolite by capillary electrophoresis.

A capillary electrophoresis method was developed and validated for the first time for the analysis of clopidogrel and its carboxylic acid metabolite. Prior to method optimization, the pH dependence of effective mobility of both compounds was determined in order to define the initial pH of the running buffer. The optimized method demonstrated to be selective, and linear in the concentration range of 2-100 microM for both compounds. The method limits of detection and quantification were, respectively, 1.2 and 3.7 microM for clopidogrel and 1.1 and 3.2 microM for the carboxylic acid metabolite. Moreover, method validation demonstrated acceptable results for method repeatability (RSD<7%), intermediate precision (RSD<7%) and accuracy (85-96%) and is suitable for the quantitative analysis of clopidogrel and its metabolite in serum samples. The validated method was also applied to the determination of the kinetic parameters of the enzymatic hydrolysis of clopidogrel. An apparent K(m) of 145+/-30 microM and V(max) of 0.4, 1.5 and 3.4 microM/min, respectively for the enzyme concentrations 1.0, 2.0 and 4.0 U/ml, were obtained.