Quantitative determination of BMS186716, a thiol compound, in dog plasma by high-performance liquid chromatography–positive ion electrospray mass spectrometry after formation of the methyl acrylate adduct

doi:10.1016/S0378-4347(97)00044-3

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 693, Issue 1, 23 May 1997, Pages 109-116

https://doi.org/10.1016/S0378-4347(97)00044-3 Get rights and content

Abstract

As it is extremely unstable in blood, the thiol compound BMS186716 was stabilized by the addition of methyl acrylate (MA) to blood samples. The blood samples were then kept in ice for 10–15 min for completion of the Michael addition reaction to occur between the thiol group of BMS186716 and MA, after which the plasma was separated by centrifugation under refrigeration. For sample analysis, the standard and quality control samples were prepared by spiking blank plasma with the BMS186716–MA adduct. After addition of the internal standard, BMS188035–MA, each sample was acidified with HCl and then extracted with methyl tert.-butyl ether. Each reconstituted extract was injected into a high-performance liquid chromatography–positive ion electrospray ionization mass spectrometric system. The electrospray condition was chosen to enhance the [M+NH₄]⁺ signal at the expense of the [M+H]⁺ signal. Monitoring the [M+NH₄]⁺ signal, a lower limit of quantitation of 2.5 ng/ml was achieved, with 0.5 ml plasma. We have thus shown that a sulfhydryl compound (BMS186716) in blood can successfully be stabilized by reacting it with MA and that the adduct produced is adequately stable in blood and plasma to allow the development of a rugged quantitative bioanalytical method.

Introduction

BMS186716 (Fig. 1) is a novel dual metalloprotease inhibitor that is being developed for the treatment of hypertension and congestive heart failure. A high-performance liquid chromatography–positive ion electrospray mass spectrometry (HPLC–MS) method for the quantitative determination of BMS186716 in dog plasma has been developed to support pharmacokinetic, bioavailability and toxicokinetic studies in dog. The objective of this paper is to describe the development of the bioanalytical method and to present the validation data.

Due to the presence of a free sulfhydryl group, BMS186716 is extremely unstable in both blood and plasma; therefore, it was necessary to stabilize the compound in blood and plasma by reacting the sulfhydryl group with methyl acrylate (MA) to form the stable Michael addition product, BMS186716–MA adduct. The use of acrylic acid esters, such as MA, for the stabilization of thiol compounds has been described previously 1, 2. The advantage of using MA over N-ethylmaleimide (NEM) for the stabilization of thiol compounds has been demonstrated previously [2]. HPLC coupled with electrospray MS, both in the positive and negative ion mode, is a proven technique for the rapid and sensitive quantitative determination of drugs and metabolites in biological matrices 3, 4, 5, 6, 7, 8.

Section snippets

Reagents and chemicals

BMS186716 and BMS188035 (the internal standard) are characterized products of Bristol-Myers Squibb Pharmaceutical Research Institute. Glacial acetic (Mallinckrodt brand), methyl tert.-butyl ether (HPLC grade), and concentrated hydrochloric acid (Mallinckrodt brand) were purchased from Baxter Scientific Products (McGaw Park, IL, USA). Laboratory-deionized water, further purified with a Milli-Q water purifying system (Millipore, Bedford, MA, USA), was used. Ammonium acetate (ACS grade) was from

Results and discussion

The reaction of MA with BMS186716 is depicted in Fig. 2. The thiol of BMS186716 undergoes the Michael addition reaction across the conjugated carbon–carbon double bond of MA to give BMS186716–MA. The reaction of BMS186716 with MA in 0.01 M disodium phosphate buffer, as described in Section 2.4, was found to be complete after 10 min at room temperature. No BMS186716 was detected after the 10 min reaction, and there was no increase in the amount of BMS186716–MA produced as the reaction period was

Conclusion

We have shown that a sulfhydryl compound (BMS186716) in blood can successfully be stabilized by reacting it with MA and that the adduct produced is adequately stable in blood and plasma to allow the development of a rugged quantitative bioanalytical method. The electrospray ionization mass spectra of the adduct, both in the positive and negative ion mode, can be easily changed by simply raising the orifice voltage to bring about CID before entering the single quadrupole mass analyzer. For the

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