Simultaneous Quantification and Pharmacokinetic Study of Five Homologs of Dalbavancin in Rat Plasma Using UHPLC-MS/MS

Abstract

Dalbavancin is a novel semisynthetic glycopeptide antibiotic that comprises multiple homologs and isomers of similar polarities. However, pharmacokinetic studies have only analyzed the primary components of dalbavancin, namely B0 and B1. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to simultaneously determinate and investigate the five homologous components of dalbavancin, namely, A0, A1, B0, B1, and B2, in rat plasma. In this method, methanol was used to precipitate plasma, and a triple-bonded alkyl chromatographic column was used for molecule separation, using 0.1% formic acid-acetonitrile as the mobile phase for gradient elution. Targeted homologs were analyzed by a triple quadrupole mass spectrometer using positive electrospray ionization in multiple reaction monitoring mode. The linearity range was 50–2500 ng/mL with a high correlation coefficient (r2 > 0.998). This method was successfully applied in the pharmacokinetic analysis of dalbavancin hydrochloride to investigate dalbavancin components in rats.

Validation and Clinical Application of a New Liquid Chromatography Coupled to Mass Spectrometry (HPLC-MS) Method for Dalbavancin Quantification in Human Plasma

“…The relatively low extraction efficiency could be partially explained by the extensive binding of DBV to plasma proteins. Other previous works which described DBV PK, using the LC-MS/MS technique for quantification, used acetonitrile for protein precipitation and did not report all the extraction efficiency and recovery data [12,15,22], while another recent work evidenced poor recovery by using acetonitrile as a precipitating solution, in perfect accordance with our results [23]. In detail, only Zhu et al [23] reported better recoveries (nearly 75%) with MeOH as a precipitating solution, while Alebich-Kolbah et al [21] chose to avoid protein precipitation, using an extreme dilution for sample preparation; nevertheless, this approach is viable only with instruments with a very high sensitivity and, moreover, could lead to problems in the long term due to the effect of remaining proteins in the final extract.…”

“…Other previous works which described DBV PK, using the LC-MS/MS technique for quantification, used acetonitrile for protein precipitation and did not report all the extraction efficiency and recovery data [12,15,22], while another recent work evidenced poor recovery by using acetonitrile as a precipitating solution, in perfect accordance with our results [23]. In detail, only Zhu et al [23] reported better recoveries (nearly 75%) with MeOH as a precipitating solution, while Alebich-Kolbah et al [21] chose to avoid protein precipitation, using an extreme dilution for sample preparation; nevertheless, this approach is viable only with instruments with a very high sensitivity and, moreover, could lead to problems in the long term due to the effect of remaining proteins in the final extract. Concerning the present study, the reproducibility of the extraction efficiency with ACN-: MeOH indicated that the impact on the analytical result remained acceptable, according to EMA and FDA guidelines, since the calibration curve and QC samples were prepared in the same matrix as the real-life specimens.…”

“…Compared with previous methods [12,15,[21][22][23], the one proposed in this work provides an extremely cheap and reliable alternative, being based on HPLC and single-MS detection, without the use of labeled IS or unavailable analogues, but also provides a very thorough description of the validation process, with particular attention to the determination of the variability in extraction efficiency and matrix effect. Obviously, the drawback of the use of HPLC is a generally longer runtime for each injection, but this drawback is well balanced with the ruggedness of the validation process in human plasma and the potential for wider use in laboratories which lack UHPLC-MS/MS instrumentation.…”

“…Nevertheless, several difficulties related to the quantification of DBV in plasma samples have been previously reported: in fact, it is well known that DBV shows extensive plasma protein binding (approximately 93%) that may cause drug co-precipitation during the extraction procedure; moreover, in the same work, a non-specific binding (NSB) to plastics has been reported [21]. Moreover, the currently published methods for DBV quantification are based on the use of highly expensive platforms and reagents, such as Ultra-High-Performance Liquid Chromatography (UHPLC) and tandem mass spectrometry (MS/MS), using isotope-labeled internal standards or analogues that are not commercially available [12,15,[21][22][23]. On the other hand, some of these methods were only validated on rat plasma [22,23] and/or their described validation process lacked recovery or matrix effect data [12,15,22], which can be extremely critical for the ruggedness of a method, particularly considering a molecule such as DBV.…”

“…Moreover, the currently published methods for DBV quantification are based on the use of highly expensive platforms and reagents, such as Ultra-High-Performance Liquid Chromatography (UHPLC) and tandem mass spectrometry (MS/MS), using isotope-labeled internal standards or analogues that are not commercially available [12,15,[21][22][23]. On the other hand, some of these methods were only validated on rat plasma [22,23] and/or their described validation process lacked recovery or matrix effect data [12,15,22], which can be extremely critical for the ruggedness of a method, particularly considering a molecule such as DBV.…”