Sensitive assay for measurement of volatile borneol, isoborneol, and the metabolite camphor in rat pharmacokinetic study of Borneolum (Bingpian) and Borneolum syntheticum (synthetic Bingpian)

Abstract

Aim: Both Borneolum (Chinese name Bingpian; dextrorotatory borneol) and Borneolum syntheticum (synthetic Bingpian; a mixture of optically inactive borneol and isoborneol) have been used for medicinal purposes in Chinese traditional medicine. The aim of this study was to develop a sensitive assay for measuring volatile ingredients borneol, isoborneol, and their metabolite camphor in pharmacokinetic study of Bingpian.

Methods: Rat plasma samples were prepared using liquid-liquid microextraction: 70 μL of plasma sample (containing 125 nmol/L naphthalene as the internal standard) was extracted with 35 μL of n-hexane. The resulting n-hexane extract (20 μL) was introduced into a gas chromatography/mass spectrometry system using programmable temperature vaporizing-based large-volume injection. The assay was validated to demonstrate its reliability for the intended use. Using this assay, pharmacokinetic studies of Bingpian, synthetic Bingpian, and Fufang-Danshen tablets (containing synthetic Bingpian) were conducted in rats.

Results: The extraction efficiency for the analytes and the internal standard from plasma was almost constant with decrease in n-hexane-to-plasma volume ratio, thus enabling a small volume of extracting solvent to be used for sample preparation, and enhancing the assay sensitivity. The lower quantification limit for measuring borneol, isoborneol, and camphor in plasma was 0.98 nmol/L, which was 33-330 times more sensitive than those reported earlier for Bingpian and synthetic Bingpian. The applicability of the miniaturized liquid-liquid extraction technique could be extended to measure other volatile and nonvolatile medicinal compounds in biomatrices, which can be predicted according to the analytes' octanol/water distribution coefficient (logD) and acid dissociation constant (pKa).

Conclusion: This assay is sensitive, accurate and free of matrix effects, and can be applied to pharmacokinetic studies of Bingpian, synthetic Bingpian, and Bingpian-containing herbal products.

Figure 1

Chemical structures of borneol, isoborneol, camphor, naphthalene, quercetin, ginkgolide A, orcinol glucoside, tanshinol, and cinnamic acid.

Figure 2

EI-MS spectra of borneol, isoborneol, camphor, and the internal standard naphthalene. The GC separation was achieved for these compounds.

Figure 3

Comparative GC-MS chromatograms for the simultaneous measurement of borneol (bp, 212–214 °C), isoborneol (212–214 °C), camphor (204 °C), and naphthalene (218 °C) in either n-hexane (69 °C) or n-pentane (36 °C) under different PTV modes with various injection volumes. The signal intensities (peak areas) of the test compounds increased significantly as the injection volume increased from 2 to 20 μL for n-hexane and from 2 to 50 μL for n-pentane, but the noise levels (measured at m/z 95 and m/z 128) did not change substantially.

Figure 4

Correlations between the organic solvent-plasma volume ratio (▵ or ▴) and extraction efficiency (○ or •) for various compounds. The nonvolatile compounds quercetin, ginkgolide A, orcinol glucoside, tanshinol, and cinnamic acid were measured by LC-MS/MS. Chromatographic separation was achieved using a Phenomenex Gemini 5 μm C18 column (50×2.0 mm id; Torrance, CA, USA). The water/acetonitrile/formic acid-based mobile phases were delivered at 0.3 mL/min for a binary pulse gradient elution. The precursor-to-product ion pairs m/z 407→351, 301→151, 331→123, 197→135, and 147→103 in the negative ESI ion mode were used for selected reaction monitoring of ginkgolide A, quercetin, orcinol glucoside, tanshinol, and cinnamic acid, respectively.

Figure 5

Rat plasma concentration-time profiles of borneol, isoborneol, and their metabolite camphor after an oral dose of Bingpian (20 mg borneol/kg), synthetic Bingpian (20 mg borneol+isoborneol/kg), or Fufang-Danshen tablets (containing synthetic Bingpian; 20 mg borneol+isoborneol/kg) or an intravenous dose of Bingpian (5 mg borneol/kg) or synthetic Bingpian (5 mg borneol+isoborneol/kg).