Simultaneous determination of losartan, EXP-3174 and hydrochlorothiazide in plasma via fully automated 96-well-format-based solid-phase extraction and liquid chromatography–negative electrospray tandem mass spectrometry

Abstract

An automated, sensitive and high-throughput liquid chromatographic/electrospray tandem mass spectrometric (LC-MS/MS) assay was developed for the simultaneous determination of losartan (LOS), its major circulating metabolite EXP-3174 and hydrochlorothiazide (HCTZ) in human plasma. LOS and HCTZ coexist in the same drug formulation, and this is the first method that enables the simultaneous determination of both drugs along with the active metabolite of LOS. Since these drugs have different physicochemical properties, the employment of a liquid-liquid extraction (LLE) protocol was precluded. A fully automated solid-phase extraction (SPE) protocol, based on 96-well format plates, was used to isolate these compounds and furosemide (internal standard, IS) from plasma. Washing and elution steps were amended accordingly in order to minimize any matrix effect from components of the plasma without reducing the elution of the molecules of interest. The compounds were eluted from a C18 column and detected with an API 3000 triple-quadrupole mass spectrometer using negative electrospray ionization and multiple reaction monitoring (MRM). The assay was linear over the range 1.00-400 ng/mL for LOS and EXP-3174 and 0.500-200 ng/mL for HCTZ, respectively, when 200 microl of plasma was used in the extraction. The overall intra- and interassay variations were within acceptance limits. The analysis time for each sample was 4 min, and more than 300 samples could be analyzed in one day by running the system overnight. The assay was simple, highly sensitive, selective, precise, fast, and it enables the reliable determination of LOS, EXP-3174 and HCTZ in pharmacokinetic or bioequivalence studies after per os administration of a single tablet containing both drugs.

Difficult-to-control arterial hypertension or uncooperative patients? The assessment of serum antihypertensive drug levels to differentiate non-responsiveness from non-adherence to recommended therapy

“…9,10 Prior to the chromatographic separation, the serum samples were prepared by liquid-liquid extraction using a mixture of ethyl acetate and dichloromethane. The chromatographic separation was performed on a reversed-phase column with a gradient flow of the mobile phase (0.05 M formic acid and acetonitrile).…”

Simultaneous analysis of losartan, its active metabolite, and hydrochlorothiazide in human plasma by a UPLC-MS/MS method

“…Kolocouri et al 45 used an automated multiprobe work station for liquid transfer steps during 96-well-based SPE after precipitation of plasma proteins with acetonitrile using 200 µ L plasma samples. Although this method is well suited for high throughput applications, this facility may not be available in all labs.…”

“…Thus, to overcome these limitations and at the same time to achieve higher sensitivity for the analytes we modified the reported procedure. 7 As all three analytes have high protein binding ( ≥68%), the plasma proteins were precipitated with 5% (v/v) ortho-phosphoric acid instead of acetonitrile 45 and use of ammonia solution 7 before loading the sample on the HLB cartridge. Further, the washing and elution steps were critically optimized without compromising the reproducibility and the recovery of all three analytes.…”

Serum drug levels to diagnose non-adherence in acute decompensated heart failure

“…10,11 ). Prior to the chromatographic separation, the serum samples were prepared by liquid-liquid extraction using a mixture of ethyl acetate and dichloromethane.…”