Nonlinear calibrations on the assay of dilitiazem and two of its metabolites from plasma samples by means of liquid chromatography and ESI/MS² detection: application to a bioequivalence study

Abstract

The assay of diltiazem (DLTZ) and its active metabolites desacetyldiltiazem (DAcD) and desmethyldiltiazem (DMeD) in plasma samples was achieved by means of an HPLC/(ESI)MS(2) method. The diastereoisomer of diltiazem, namely {(2R,3S)-5-[2-(dimethylamino)ethyl]-2-(4-methoxyphenyl)-4-oxo-2,3,4,5-tetrahydro-1,5-benzothiazepin-3-yl acetate} was used as internal standard (IS). Sample preparation was based on protein precipitation by means of organic solvent addition (acetonitrile). The isocratic elution based on a reversed-phase mechanism allows good separation of the analytes within 15 min. Atmospheric pressure electrospray ionization was used. All analytes were monitored in the MS(2)-MRM mode. A fragmentation schema is proposed for the target compounds. As the method was designed for bioequivalence purposes, a full validation procedure was considered. On validation, nonlinear calibrations were observed. Consequently, concentration intervals requiring nonlinear calibrations are discussed. Low limits of quantification in the 0.6-1 ng/mL concentration range were obtained. The analytical method was successfully applied to a single dose (120 mg), open-label, randomized, two-period, two-sequence, crossover bioequivalence study of two commercially available solid oral dosage pharmaceutical formulations (tablets).

Applicability of nonlinear calibration regression for quantitative determination of parent and metabolite(s) in bioequivalence assessment

“…In this tandem mass spectrometric method involving MRM mode with electrospray ionization, nonlinearity prompted the use of log-log regression to better fit the concentration data of the parent compound, diltiazem. Nevertheless, log-log regression also appeared to provide adequate justification for the two metabolites, namely desacetyldiltiazem and desmethydiltiazem (Georgita et al, 2008). While ordinarily it would appear a deviation from the norm of applying linear regression of the calibration curve data, the review of the data amply supports the principle of nonlinear calibration (Georgita et al, 2008).…”

“…Nevertheless, log-log regression also appeared to provide adequate justification for the two metabolites, namely desacetyldiltiazem and desmethydiltiazem (Georgita et al, 2008). While ordinarily it would appear a deviation from the norm of applying linear regression of the calibration curve data, the review of the data amply supports the principle of nonlinear calibration (Georgita et al, 2008). The authors had a choice of creating different acquisition intervals for individual peaks of interest or using a common wider acquisition window for all peaks of interest.…”

“…Some recent select examples are provided in Table 1-the list includes compounds belonging to various therapeutic areas. However, the recent report of Georgita et al (2008) highlights the need for other options outside of linear regression for quantification of parent and metabolite(s) in the assessment of bioequivalence. In this tandem mass spectrometric method involving MRM mode with electrospray ionization, nonlinearity prompted the use of log-log regression to better fit the concentration data of the parent compound, diltiazem.…”

“…The authors had a choice of creating different acquisition intervals for individual peaks of interest or using a common wider acquisition window for all peaks of interest. The choice of wider isolation was preferred over the other choice and this necessitated the use of log-log calibrations for all peaks of interest (Georgita et al, 2008). It should be noted that the work of Georgita et al (2008) eloquently captures all required elements to unequivocally establish the choice of log-log regression over the traditional use of dynamic linear range of quantitation.…”

Applicability of LC/MS/MS assay for 6‐methoxy‐2‐napthylacetic acid to support the bioequivalence study of nabumetone—comments on the research work of Patel et al. (2008)

“…Applicability of LC/MS/MS assay for 6-methoxy-2-napthylacetic acid to support the bioequivalence study of nabumetonecomments on the research work of Patel et al (2008) To the EditorIn recent years, many publications have appeared in BMC that utilize state of the art laboratory tools for development and validation of assays for the applicability to bioequivalence studies in human volunteers (Georgita et al, 2008;Patel et al, 2008;Medvedovici et al, 2008;Sora et al, 2007;Nirogi et al, 2006). Interestingly, all of these published assays support both parent and/or active metabolite(s) exposure measurements to facilitate pharmacokinetic characterization and/or bioequivalence assessment of the derived parameters between test and reference products.…”