Florin Albu

Large volume injection of 1-octanol as sample diluent in reversed phase liquid chromatography: Application in bioanalysis for assaying of indapamide in whole blood

Large volume injection of samples in strong diluents immiscible with the mobile phases used in reversed phase liquid chromatography (RPLC) has been recently introduced in practice. In the present work, the potential of the technique has been evaluated for bioanalytical applications. The process consists of the liquid-liquid extraction of indapamide from whole blood into 1-octanol, followed by the direct injection from the organic layer into the LC. Detection was made through negative electrospray ionization (ESI) and tandem mass spectrometry (MS(2)). The method was developed, validated, and successfully applied to a large number of samples in two bioequivalence studies designed for indapamide 1.5mg sustained release and 2.5mg immediate release pharmaceutical formulations. The performance of the analytical method is discussed based on data resulting from the validation procedure and the completion of the bioequivalence studies.

Retention Behavior of Metformin and Related Impurities in Ion‐Pairing Liquid Chromatography

Abstract The ion‐pair (IP) mechanism was applied to achieve HPLC‐diode‐array detector (DAD) separation and determination of metformin and five related impurities (cyanoguanidine, 1‐methylbiguanidine, melamine, N,N‐dimethylmelamine, and 4,6‐diamino‐1,3,5‐triazin‐2‐yl guanidine). Sodium octanesulphonate (0.010 mol/L) at pH = 3 was used as an ion pair agent in the aqueous component of the mobile phase. Isocratic elution with 80% acetonitrile and 20% aqueous component was applied with a flow rate of 1.2 mL/min. A double end‐capped, 5 µm particle size Inertsil ODS‐2 stationary phase (250 mm length, 4.6 mm internal diameter column) was used. Limit of quantitation (LOQ), as absolute amount of analytes loaded to the column, in the 35–250 pg interval were obtained using detection at 218 nm. Retention data were used in estimating the thermodynamic parameters (enthalpy and entropy) corresponding to the mobile/stationary phase transfer. Correlations between experimental data and data predicted by means of basicity constant, pH, and water/octanol partition coefficient were also achieved.

Analytical issues in HPLC/MS/MS simultaneous assay of furosemide, spironolactone and canrenone in human plasma samples

A new sensitive HPLC/MS/MS method for simultaneous determination of furosemide, spironolactone and canrenone in human plasma samples is presented. Electrospray ionization source (ESI) has been used. The tandem MS detection was performed under MRM conditions, in the negative ion mode for furosemide and indapamide (internal standard 1) and in the positive ion mode for spironolactone, canrenone and nitrazepam (internal standard 2). A simple plasma protein precipitation with acetonitrile was used as sample preparation technique. The chromatographic separation was carried out under the reversed phase mechanism, on a 250 mm length column packed with octadecyl modified silicagel and thermostated at 35 degrees C. The column was operated under isocratic conditions (3:7 aqueous 0.1% formic acid and methanol, v/v) at a flow rate of 0.8 mL/min. Quantitation intervals of 20-1600 ng/mL for furosemide and 2-100 ng/mL for spironolactone and canrenone have been concluded through validation. Precision and accuracy were situated within the accepted thresholds (maximum 15% relative standard deviation and +/-15% percentage bias). The most sensitive aspects relating to the analytical method development and validation were highlighted and critically assessed in order to reach an objective opinion about the real performances and inherent applicability of the method in bioanalysis.

Structure–Retention Correlation of Some Oxicam Drugs in Reversed‐Phase Liquid Chromatography

Abstract The main parameters related to the retention behavior of some oxicam drugs (meloxicam, piroxicam, and tenoxicam) in reversed‐phase liquid chromatography (RP‐LC) processes using a monolithic column were studied under isocratic elution conditions. The hydrophobicity parameter (octanol/water partition coefficient) obtained by extrapolation of the capacity factor—methanol content of the mobile phase relationship did not fit the well‐known dependence on pH. This fact might be assigned to the keto–enolic equilibrium that does not behave like common acids. The dissociation constants for these oxicams were estimated from the retention dependence on the organic modifier content (methanol) in the mobile phase and pH of aqueous components (interval 2–7). Two corrections were applied to the hydrophobicity parameter of analytes, taking into consideration the pH of aqueous component in the mobile phase, and hydrophobicity parameter of the stationary phase. The results were compared to other experimental data and data predicted by the fragment methodology.

Greener bioanalytical approach for LC/MS-MS assay of enalapril and enalaprilat in human plasma with total replacement of acetonitrile throughout all analytical stages.

Green bioanalytical approaches are oriented toward minimization or elimination of hazardous chemicals associated to bioanalytical applications. LC/MS-MS assay of enalapril and enalaprilat in human plasma was achieved by elimination of acetonitrile from both sample preparation and chromatographic separation stages. Protein precipitation (PP) by acetonitrile addition was replaced by liquid-liquid extraction (LLE) in 1-octanol followed by direct large volume injection of the organic layer in the chromatographic column operated under reversed phase (RP) separation mechanism. At the mean time, acetonitrile used as organic modifier in the mobile phase was successfully replaced by a mixture of propylene carbonate/ethanol (7/3, v/v). Three analytical alternatives ((I) acetonitrile PP+acetonitrile based chromatographic elution; (II) 1-octanol LLE+acetonitrile based chromatographic elution; (III) 1-octanol LLE+propylene carbonate/ethanol based chromatographic elution) were validated and the quality characteristics were compared. Comparison between these alternative analytical approaches was also based on results obtained on incurred samples taken during a bioequivalence study, through application of the Bland-Altman procedure.

Assay of free captopril in human plasma as monobromobimane derivative, using RPLC/(+)ESI/MS/MS: validation aspects and bioequivalence evaluation

A sensitive method for determination of free captopril as monobromobimane derivative in plasma samples is discussed. The internal standard (IS) was 5-methoxy-1H-benzimidazole-2-thiol. Derivatization with monobromobimane immediately after blood collection and plasma preparation prevents oxidation of captopril to the corresponding disulfide compound and enhances the ionization yield. Consequently, derivatization enhances sample stability and detection sensitivity. Addition of the internal standard was made immediately after plasma preparation. The internal standard was also derivatized by monobromobimane, as it contains a thiol functional group. Preparation of plasma samples containing captopril and IS derivatives was based upon protein precipitation through addition of acetonitrile, in a volumetric ratio 1:2. The reversed-phase liquid chromatographic separation was achieved on a rapid resolution cartridge Zorbax SB-C(18), monitored through positive electrospray ionization and tandem MS detection using the multiple-reaction monitoring mode. Transitions were 408-362 amu for the captopril derivative and 371-260 amu for the internal standard derivative. The kinetics of captopril oxidation to the corresponding disulfide compound in plasma matrix was also studied using the proposed method. A linear log-log calibration was obtained over the concentration interval 2.5-750 ng/mL. A low limit of quantitation in the 2.5 ng/mL range was obtained. The analytical method was fully validated and successfully applied in a three-way, three-period, single-dose (50 mg), block-randomized bioequivalence study for two pharmaceutical formulations (captopril LPH 25 and 50 mg) against the comparator Capoten 50 mg.

Solvent and salting effects on sample preparation for the determination of fenofibric acid in human plasma by HPLC-DAD

Abstract The solvent and salting effects induced on the sample preparation procedure applied to plasma samples containing fenofibric acid and 4-chlorophenyl-4′-hydroxyphenyl methanone (internal standard) are evaluated. Sodium chloride addition during a deproteinization step using both methanol and phosphoric acid influences the recovery of the analytes as well as the selectivity of the process. The chromatographic method allows high sample volume injection (500 μl) with the focusing of both analytes in the stationary phase. The synthesized high porosity octadecylsilica material allows a fast elution gradient at 4 ml/min flow-rate and a complete analysis within 7 min. UV-detection is made at 295 nm and quantitation limit in the 20 ng/ml concentration level can be achieved. The method can be successfully applied for bioequivalence studies on fenofibrate, administrated as prodrug (fenofibric acid represents its main active metabolite) in pharmaceutical formulations. The main parameters used in studying the retention behavior of the internal standard and FEFA were also estimated.

Assay at low ppm level of dimethyl sulfate in starting materials for API synthesis using derivatization in ionic liquid media and LC–MS

Dimethyl sulfate (DMS) is frequently used in pharmaceutical manufacturing processes as an alkylating agent. Trace levels of DMS in drug substances should be carefully monitored since the compound can become an impurity which is genotoxic in nature. Derivatization of DMS with dibenzazepine leads to formation of the N-methyl derivative, which can be retained on a reversed phase column and subsequently separated from other potential impurities. Such derivatization occurs relatively slowly. However, it can be substantially speed up if ionic liquids are used as reaction media. In this paper we report the use of 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide (IL1) and 1-butyl-4-methylpyridinium tetrafluoroborate (IL2) as reaction media for the derivatization of DMS with dibenzazepine. It was determined that the stoichiometry between the substrate and DMS may be 1:1 or 2:1, in relation with the nature of the reaction media. An (+)ESI-MS/MS approach was used for quantitation of the derivatized product. Alternatively, DMS derivatization may be carried out with pyridine in acetonitrile (ACN). The N-methylpyridinium derivative was separated by hydrophilic interaction liquid chromatography (HILIC) and detected through (+)ESI-MS (in the SIM mode). In both cases, a limit of quantitation (LOQ) of 0.05 μg/ml DMS was achievable, with a linearity range up to 10 μg/ml. Both analytical alternatives were applied to assay DMS in 4-(2-methoxyethyl)phenol, which is used as a starting material in the synthesis of metoprolol.

HANDLING DRAWBACKS OF MASS SPECTROMETRIC DETECTION COUPLED TO LIQUID CHROMATOGRAPHY IN BIOANALYSIS

Hyphenation of mass spectrometry (MS) to liquid chromatography (LC) represents a powerful tool for qualitative and quantitative characterization of target compounds in very complex matrixes of biological origins. In spite of many advantages due to recent advances and innovations in the area of instrumentation and dedicated software support, some difficulties are still encountered in its current applications. The large variety of functional principles and technical solutions applied for hyphenation of the two techniques, for ion sources, ion extraction and focusing, mass analysis, and ion counting makes it more difficult to obtain perfect agreement between the intrinsic characteristics of the laboratory-available instrumentation and the declared goals of specific determination. This review covers a part of the literature data dealing with the shortcomings of LC/MS in bioanalysis. The following topics are discussed: structural identification and confirmation in LC/MS; precision of the instrumental response over short and long term periods; non-linear response functions; adduct formation in atmospheric pressure ion sources; and carryover effects. Most of the problems arising in LC/MS are related to phenomena occurring during ionization. Obviously, the structural characteristics of the analyzed compounds play an important role, although the principles of ionization within the source and the supporting technical solutions and constructive designs add their own particular features. The complex influence of residual sample matrixes over ionization yields of target compounds and internal standards needs to be studied through proper experimental procedures, in order to control both precision and instrumental response function in analysis of biological samples.

On-Line SPE on Restricted Access Adsorbent for HPLC-MS/MS Analysis of Felodipine in Plasma Samples

Direct plasma loading on a LiChrospher ADS C18 cartridge on-line coupled to an analytical Zorbax XDB C18 column was used to analyze felodipine by means of positive atmospheric pressure chemical ionization (APCI) tandem mass spectrometric (MS/MS) detection. Appropriate sensitivity, accuracy, and precision were obtained using an ion trap mass analyzer operated in a multiple reaction monitoring (MRM) mode. Diethyl-4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate) was used as an internal standard. The method was fully validated. The method was successfully applied to a pilot bioequivalence study made on extended release oral dosage forms containing 10 mg of felodipine, under fed and fasting conditions.