Bioanalytical methods for the detection of duloxetine and thioctic acid in plasma using ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS)

Abstract

Duloxetine and thioctic acid (TA) are standard drugs for treating diabetic neuropathy, a primary complication associated with diabetes. In this study, ultra performance liquid chromatography coupled with tandem mass spectrometry methods was successfully developed and validated for quantifying duloxetine and TA in biological samples. The protein precipitation method was used to extract duloxetine, TA and their internal standards from beagle dog plasma. A Hypersil Gold C18 column (150 × 2.1 mm, 1.9 μm) was used for the experiment. Isocratic elution with 0.1% formic acid in acetonitrile (A) and 0.1% formic acid (B) was used for duloxetine, whereas a gradient elution with 0.03% acetic acid (A) and acetonitrile (B) was used for TA. The validated parameters included linearity, sensitivity, accuracy, precision, selectivity, matrix effect, stability, and recovery under different conditions. The linear ranges of the calibration curves for duloxetine and TA were 5-800 ng/mL and 5-1,000 ng/mL, respectively. An intra- and inter-run precision of ± 15% can be observed in all quality control samples. These methods were successfully used for pharmacokinetics (PKs) studies in beagle dogs to compare PK differences in a fixed-dose combination including duloxetine and TA and co-administration of the 2 drugs.

Keywords: Duloxetine; Liquid Chromatography; Mass Spectrometry; Thioctic Acid.

Figure 1. Mass spectra of (A) duloxetine and (B) internal standard (duloxetine-d₃) in ESI positive mode, and (C) thioctic acid and (D) its internal standard (thioctic acid-d₅) in ESI negative mode.

ESI, electrospray ionization.

Figure 2. Representative chromatograms of double blank beagle dog plasma containing (A) duloxetine and (B) its LLOQ samples, and (C) thioctic acid and (D) its LLOQ samples.

LLOQ, lower limit of quantification; NL, normalization level; TIC, total ion chromatogram; MS, mass spectrometry; ICIS, interactive chemical information system; RT, retention time; MRM, multiple reaction monitoring; ESI, electrospray ionization; SRM, selected reaction monitoring.

Figure 3. Representative chromatograms of (A) duloxetine and IS (duloxetine-d₃), and (B) thioctic acid and IS (thioctic acid-d₅) in beagle dog plasma after administration of a fixed-dose combination tablet of duloxetine and thioctic acid.

IS, internal standard; NL, normalization level; TIC, total ion chromatogram; MS, mass spectrometry; ICIS, interactive chemical information system; RT, retention time; MRM, multiple reaction monitoring; ESI, electrospray ionization; SRM, selected reaction monitoring.

Figure 4. Plasma (A) duloxetine and (B) thioctic acid concentration–time profiles after administration of duloxetine (60 mg) and thioctic acid (480 mg) as separate doses (reference groups, pink circle), or as a fixed-dose combination tablet (test group, purple circle). Error bars indicate the standard deviation.