Development of a Rapid and Simultaneous Detection Method for Buprenorphine, Norbuprenorphine and Naloxone in Human Plasma Using Ultra‐high Performance Liquid Chromatography‐tandem Mass Spectrometer with Solid‐phase Extraction

Abstract

A simultaneous method was successfully established and validated for the separation and determination of buprenorphine (BP), its primary metabolite, nor-buprenorphine (NBP) and a proposed co-formulate, naloxone (NLX) in human plasma. The method used buprenorphine-d 4 (BP-D 4 ), nor-buprenorphine-d 3 (NBP-D 3 ), naltrexone (NTX) as internal standards (ISs). 100 µL of plasma sample fortified with the ISs was cleaned up by solid-phase extraction (SPE), and was then separated on a Waters Acquity TM BEH C 18 column with gradient elution using methanol and water (containing 0.2% formic) at a flow rate of 0.25 mL•min -1 . The mass spectrometer was used for detection and was operated in the positive electrospray ionization with multiple reaction monitoring (MRM) mode. The three compounds were effectively separated in 5 min. The linear ranges of the compounds were 0.1-25, 0.25-25 and 0.05-25 ng•mL -1 for BP, NBP and NLX, respectively, with r≥0.9935. The method had high sensitivity (the limits of detection were 0.02, 0.1 and 0.01 ng•mL -1 for BP, NBP and NLX, respectively) and high recoveries (≥97.6%). The result was shown to be linear and satisfactorily met current acceptance criteria for validation of bioanalytical method: intra and inter assay precisions within the required limits of ≤25% RSD. The LOQs fulfilled the LOQ requirements: precision≤25% RSD, and was fully validated according to the State Food and Drug Administration (SFDA) regulations. The results demonstrated that ultra-high performance liquid chromatographytandem mass spectrometer (UPLC-MS/MS) with SPE was a powerful detection tool and contributed to pharmaceutical analysis in biological matrices.

A sensitive, simple and rapid HPLC–MS/MS method for simultaneous quantification of buprenorpine and its N-dealkylated metabolite norbuprenorphine in human plasma

“…Reported methods with more advantageous instruments like UPLC applied, have at least 7.8 min run time to simultaneously quantify BUP and NBUP, which are not ideally fit to analyzing pharmacokinetic samples in a high-throughput manner [23] . In this developed method, a good chromatographic separation of BUP and NBUP was achieved within 4.1 min by using a reverse-phase (C18) column in gradient mode.…”

“…Limited sensitivity also caused difficulties in the sample treatment. SPE (solid-phase extraction) was applied in many methods to clean up the plasma sample, which made the sample treatment highly cost and time consuming [23] . LLE (liquid–liquid extraction) was a simpler choice for sample treatment; however, larger sample volume together with large volume of extraction solvent was required, which was not suitable for green chemistry and made the procedures not practical in high-throughput analysis [24] .…”

“…Minimum effective concentration of the transdermal patch formulation of buprenorphine (Transtec ® ) was only 100 pg/mL in plasma and C max was only 305±117 pg/mL for 35 μg/h dose, which indicating the necessary for a highly sensitive method [6] . To date, liquid chromatography tandem mass spectrometry (LC-MS), with its advantages of high selectivity, sensitivity and accuracy, becomes a major technique for quantification of BUP and its metabolites in a wide variety of matrices, including urine [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] , [15] , plasma [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] , [24] , whole blood [9] , [10] , [11] , [25] , hair [9] , [10] , [11] , [26] , [27] , [28] , sweat [29] , meconium [30] , and breast milk [31] . Of the current available LC-MS methods applied in plasma, the lack of the requisite sensitivity and selectivity for accurate assessment of the drug concentrations is the major limitation.…”

“…Limited sensitivity in turn required larger sample volume, which is also not practical in sample analysis. In order to achieve good separation from the high baseline caused by endogenous materials, though more advantageous instruments like UPLC were applied, longer run time of LC separation is still required [16] , [17] , [18] , [19] , [20] , [21] , [22] , [23] . A simpler, economic sample treatment procedure with excellent reproducibility and high recovery is also in need to meet the requirement of high-throughput analysis.…”