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Determination of tapentadol and tapentadol-O-glucuronide in human serum samples by UPLC-MS/MS.

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2015-01-21)
Vera Hillewaert, Klaus Pusecker, Luc Sips, Tom Verhaeghe, Ronald de Vries, Manfred Langhans, Rolf Terlinden, Philip Timmerman
ABSTRACT

Tapentadol is a novel, centrally acting analgesic with 2 mechanisms of action, MOR agonism and noradrenaline (NA) reuptake inhibition in a single molecule. It is the first member of a new therapeutic class, MOR-NRI. A high throughput liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay was developed and validated for the quantitative analysis of tapentadol and its O-glucuronide metabolite in human serum. Simultaneous quantification was deemed to be challenging because of the large difference in concentrations between tapentadol and its O-glucuronide metabolite in clinical samples. Therefore, a method was established using a common processed sample, but with different injection volumes and chromatographic conditions for each analyte. Tapentadol and tapentadol-O-glucuronide were determined by protein precipitation of 0.100ml of the samples with acetonitrile. The internal standards used are D₆-tapentadol and D₆-tapentadol-O-glucuronide. The validated concentration range was 0.200-200 ng/ml (tapentadol) and 10.0-10,000 ng/ml (tapentadol-O-glucuronide). Chromatographic separation was achieved by gradient elution on a Waters Acquity UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) column, with mobile phase consisting of 0.01 M ammonium formate (adjusted to pH 4 using formic acid) (A) and methanol (B). A separate injection was done for measurement of each analyte, with a different gradient and run time. The analytes were detected by using an electrospray ion source on a triple quadrupole mass spectrometer operating in positive ionization mode. The run time was 1.6 min for tapentadol and 1.5 min for tapentadol-O-glucuronide. The high sensitivity and acceptable performance of the assay allowed its application to the analysis of serum samples in clinical trials. The validated method was used for analysis of tapentadol in over 17,000 samples.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
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Methanol, suitable for HPLC, ≥99.9%
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Methanol, anhydrous, 99.8%
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Methanol, HPLC Plus, ≥99.9%
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Methanol, suitable for HPLC, gradient grade, ≥99.9%
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Ammonium formate, eluent additive for LC-MS, LiChropur, ≥99.0%
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Methanol, ACS reagent, ≥99.8%
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Formic acid, reagent grade, ≥95%
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Acetonitrile, for HPLC, for UV, ≥99.9% (GC)
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Acetonitrile, ACS reagent, ≥99.5%
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Formic acid, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥98%
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Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
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Ammonium formate, reagent grade, 97%
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USP
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Formic acid, puriss., meets analytical specifications of DAC, FCC, 98.0-100%
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Ammonium formate, BioUltra, ≥99.0% (calc. based on dry substance, NT)
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