Stefan Udrescu

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.

Effect of large volume injection of hydrophobic solvents on the retention of less hydrophobic pharmaceutical solutes in RP‐LC

Injection of large volumes of samples in solvents other than mobile phase composition has been proved for some less hydrophobic compounds. Thus, the retention behavior of several compounds of pharmaceutical interest (isosorbide-2-nitrate, isosorbide-5-nitrate, tropicamide, pentoxifylline, and methyl p-hydroxybenzoate) was studied by using different hydrophobic solvents (n-hexane, n-heptane, or i-octane) as sample solvents. Two types of stationary phases were used: octyl and octadecyl modified silica (both of Zorbax Eclipse type). The experiments showed a linear dependence between capacity factor of each solute and sample injection volume, up to maximum volume values of about 680 microL for C8 stationary phase and 580 microL for C18 stationary phase, when the solutes are no longer retained in stationary phase. Injection of large volumes of these hydrophobic solvents is thus possible in RP-LC with a gradual reduction of retention and peak efficiency. Two major conditions are however necessary in order to apply such an injection approach: the solutes must have a proper solubility in hydrophobic solvents and meanwhile they have to be less hydrophobic than the sample solvent in order to avoid competition with solvent molecules in partitioning between mobile and stationary phases.

Use of a green (bio) solvent – limonene – as extractant and immiscible diluent for large volume injection in the RPLC-tandem MS assay of statins and related metabolites in human plasma

Limonene, considered a green solvent, was successfully used to extract simvastatin, lovastatin, and their hydroxy-acid metabolites from human plasma samples. The extraction process was followed by the direct injection of a large volume aliquot (100 μL) from the limonene layer into a Zorbax SB-C(18) Rapid Resolution chromatographic column (50 mm length × 4.6 mm i.d. × 1.8 µm d.p.), operated under gradient elution reversed-phase separation mechanism. Tandem mass spectrometry operated under the multiple reaction monitoring mode was used for detection, providing low quantitation limits in the 0.25-0.5 ng/mL concentration interval. This method was validated and used for quantitation of simvastatin and its hydroxy acid metabolite in incurred plasma samples obtained from two volunteers participating in a bioequivalence study, using lovastatin and its hydroxy analog as internal standards. The results were statistically compared with those produced by means of an alternative RPLC-tandem MS using protein precipitation with acetonitrile. The quality attributes of the two methods are comparatively discussed. The agreement between the quality characteristics of the two methods and the experimental results obtained on real samples may be considered as a consistent basis for the simultaneous use of limonene as extraction medium and injection diluent for hydrophobic compounds in bioanalytical approaches.

Large-volume injection of sample diluents not miscible with the mobile phase as an alternative approach in sample preparation for bioanalysis: an application for fenspiride bioequivalence

BACKGROUND Liquid-liquid extraction of target compounds from biological matrices followed by the injection of a large volume from the organic layer into the chromatographic column operated under reversed-phase (RP) conditions would successfully combine the selectivity and the straightforward character of the procedure in order to enhance sensitivity, compared with the usual approach of involving solvent evaporation and residue re-dissolution. Large-volume injection of samples in diluents that are not miscible with the mobile phase was recently introduced in chromatographic practice. The risk of random errors produced during the manipulation of samples is also substantially reduced. RESULTS A bioanalytical method designed for the bioequivalence of fenspiride containing pharmaceutical formulations was based on a sample preparation procedure involving extraction of the target analyte and the internal standard (trimetazidine) from alkalinized plasma samples in 1-octanol. A volume of 75 µl from the octanol layer was directly injected on a Zorbax SB C18 Rapid Resolution, 50 mm length × 4.6 mm internal diameter × 1.8 µm particle size column, with the RP separation being carried out under gradient elution conditions. Detection was made through positive ESI and MS/MS. Aspects related to method development and validation are discussed. CONCLUSIONS The bioanalytical method was successfully applied to assess bioequivalence of a modified release pharmaceutical formulation containing 80 mg fenspiride hydrochloride during two different studies carried out as single-dose administration under fasting and fed conditions (four arms), and multiple doses administration, respectively. The quality attributes assigned to the bioanalytical method, as resulting from its application to the bioequivalence studies, are highlighted and fully demonstrate that sample preparation based on large-volume injection of immiscible diluents has an increased potential for application in bioanalysis.

LARGE VOLUME INJECTION OF HEXANE SOLUTIONS IN RPLC/UV TO ENHANCE ON SENSITIVITY OF THE ASSAY OF GINKGOLIC ACIDS IN GINGKO BILOBA STANDARDIZED EXTRACTS

The sensitivity of the compendial European Pharmacopoeia RPLC/UV method for assaying residual ginkgolic acids in Ginkgo biloba standardized extracts was essentially improved by operating modifications on the sample preparation procedure, including sample injection. The acidified methanol/water solution of the Ginkgo biloba standardized extract was extracted “in situ” with n-hexane (concentration factor of 20). From the organic layer, a relatively large volume (50 µL) of the hexane solution was directly loaded to the chromatographic column. A modified gradient elution profile was used in order to force the sample solvent to elute faster than analytes, without affecting peak symmetry. The separation is obtained in 22 minutes. As the amount of the target compounds loaded to column is higher, selective UV detection at 310 nm was possible. The method was validated according to specific operating guidelines for selectivity, linearity range, quantitation limits, precision, accuracy, and robustness. A limit of quantitation of 1 ppm (five times less than the accepted maximal content threshold for ginkgolic acids in standardized G. biloba extracts) was obtained. Confirmation of the target compounds through MS2 detection and evaluation of the sensitivity afforded by such a detection system are also discussed.

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.

LC-MS/MS assay of quinapril and its metabolite quinaprilat for drug bioequivalence evaluation: prospective, concurrential and retrospective method validation

BACKGROUND The bioequivalence of two pharmaceutical formulations containing 10 mg quinapril was assessed by assaying the untransformed drug and its active metabolite quinaprilat from plasma samples. RESULTS A gradient elution liquid chromatographic separation coupled to positive atmospheric pressure electrospray ionization and tandem mass spectrometry detection was used and validated. Sample preparation is simple and uses protein precipitation through addition of an acetonitrile:methanol (8:2 v/v) mixture. The method has a run time of 6.3 min. Carvedilol was used as an internal standard. The multiple reactions monitoring mode was used for both quantitation and structural confirmation of target compounds. Linear 1/x²-weighted regressions characterize detector response function up to concentrations of 1000 ng/ml for quinapril and 2000 ng/ml for quinaprilat. Low limits of quantitation of 5 ng/ml for quinapril and 10 ng/ml for quinaprilat were found. Intra- and inter-day variability of the results were found below 15%. Long- (-20°C/6 months) and short-term (25°C/48 h) stability of analytes in plasma, as well as freeze and thaw stability (six cycles) were demonstrated. CONCLUSION The method was found to be selective, precise, accurate and robust when applied to a large number of unknown samples.