Introduction

Many important bioactive constituents of herbal medicines are volatile compounds1,2,3,4, which are commonly analyzed using hyphenated gas chromatography (GC) techniques5,6. To understand the contribution of these compounds to the medicinal effects of the herbal medicines containing them, it is important to investigate the pharmacokinetics and disposition of the volatile ingredients. However, associated pharmacokinetic (PK) studies are often hampered by assay failure in the measurement of such herbal compounds and their volatile metabolites in complex biomatrices. Liquid-liquid extraction is often used for GC-based analysis, which involves the transfer of volatile analytes from the aqueous biological sample into a water-immiscible organic solvent. Because of the column's low loading capacity (normally 1–2 μL), only a small portion of the organic extract is allowed to be introduced directly into the GC capillary column, which is unfavorable for the assay sensitivity. Conventional concentration of the organic extract under either reduced pressure or a stream of nitrogen gas can lead to serious evaporative losses of the volatile analytes and, in turn, result in unacceptable assay accuracy and the loss of sensitivity. To support trace analysis, Vogt developed a programmable temperature vaporizing (PTV) injector for large-volume injection (LVI) of an organic sample (up to 250 μL) in capillary GC, which involves a quick solvent vent technique7. Successful implementation of PTV-based LVI requires the use of an organic solvent that has a sufficiently lower boiling point (bp) than that of the analyte (typically >100 °C). Additionally, the solvent vapor needs to be minimized to avoid its substantial transfer to the vacuum system of the mass spectrometer. Furthermore, the solvent venting also needs to be stopped shortly before the completion of solvent evaporation because loss of the volatile analyte occurs primarily with the evaporation of the last few microliters of solvent8.

Borneolum (prepared from either Dryobalanops aromatica or Cinnamonum camphora) and Borneolum syntheticum (synthetically produced from turpentine oil) are used in Chinese traditional medicine under the name “Bingpian”9,10. Borneolum is dextrorotatory borneol (endo-1,7,7-trimethyl-bicyclo[2.2.1]heptan-2-ol) (Figure 1), which is a bicyclic monoterpene alcohol, whereas Borneolum syntheticum (also called synthetic Bingpian) is a mixture of optically inactive borneol and isoborneol (exo-1,7,7-trimethyl-bicyclo[2.2.1]heptan-2-ol). Bingpian and synthetic Bingpian exert medicinal actions, including restoring consciousness, removing heat, and relieving pain. Either Bingpian or synthetic Bingpian are commonly used as an ingredient in many Chinese combination herb therapies for treating cardiovascular and cerebrovascular diseases. Borneol and isoborneol have been reported to have a variety of in vitro pharmacological activities, including vasorelaxant11, anticoagulant12, neuroprotective13,14, sedative, and analgesic properties15,16. In addition, Bingpian and synthetic Bingpian have attracted much attention because of its possible enhancement of the oral bioavailability of other drugs and medicinal herbal compounds17,18, and the passage across the blood brain barrier19,20,21. Borneol can be oxidized to camphor (1,7,7-trimethylbicyclo[2.2.1]heptan-2-one), which has been observed in mice, rats, and rabbits receiving an oral dose of either Bingpian or synthetic Bingpian22. Camphor is believed to be poisonous when ingested in large quantities and can cause seizures, confusion, irritability, and neuromuscular hyperactivity23.

Figure 1
figure 1

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

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As part of our ongoing pharmacokinetic study of Fufang-Danshen-Fang24,25,26, a cardiovascular herb combination containing Salvia miltiorrhiza roots (Danshen), Panax notoginseng roots (Sanqi), and Bingpian, we developed a sensitive bioanalytical assay for the measurement of plasma concentrations of borneol, isoborneol, and their metabolite camphor. Several other assays have been reported for measurement of borneol and isoborneol in biomatrices using either GC-mass spectrometer27,28 or GC–flame ionization detector29,30,31,32,33. However, these assays lack sensitivity and/or accuracy for one of two reasons: 1) only a small portion of the organic sample extract was introduced into the GC capillary columns, or 2) the evaporative concentration of the organic extract was used for the sample clean-up. High sensitivity was achieved for our newly developed assay by using a liquid-liquid microextraction technique working in concert with an optimal PTV-based LVI of the organic extract, which led to significantly increased amounts of the analytes from the plasma sample being applied to GC-mass spectrometry-based analysis. To our knowledge, this is the first method of liquid-liquid microextraction combined with PTV-based LVI that has been developed for the measurement of plasma concentrations of volatile herbal compounds.

Materials and methods

Chemicals and materials

Analytical reference standards of (±)-borneol (bp 212–214 °C), (±)-isoborneol (212–214 °C), (±)-camphor (204 °C), naphthalene (218 °C), quercetin, ginkgolide A, tanshinol (sodiated form), and cinnamic acid were obtained from the National Institutes for Food and Drug Control (Beijing, China). Orcinol glucoside was kindly donated by Professor Jijun CHEN from Kunming Institute of Botany, the Chinese Academy of Sciences (Kunming, China). The purity of these compounds was greater than 98%. HPLC-grade organic solvents were obtained from Sinopharm Chemical Reagent Co, Ltd (Shanghai, China). HPLC-grade water was prepared with an in-house Millipore Milli-Q Integral 3 cabinet water purifying system (Bedford, MA, USA).

Bingpian (D. aromatica) and synthetic Bingpian for animal studies were obtained from Shanghai Huayu Chinese Herbs Co, Ltd (Shanghai, China). The Bingpian contained only borneol, which comprised 96.4% of the drug. The synthetic Bingpian contained both borneol and isoborneol, which comprised 62.1% and 37.6% of the drug, respectively. Fufang-Danshen tablets (Chinese SFDA ratification No Z31020478; Shanghai Leiyunshang Pharmaceutical Co, Ltd, Shanghai, China) contained Danshen, Sanqi, and synthetic Bingpian. The herbal tablets (0.32 g each) contained borneol (3.72 mg/tablet) and isoborneol (1.98 mg/tablet), which were standardized to contain ≥0.2 mg tanshinone IIA/tablet and ≥5.0 mg salvianolic acid B/tablet. Blank plasma was obtained from normal Sprague-Dawley rats receiving no drug treatment. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the Shanghai Institute of Materia Medica (Shanghai, China).

GC-MS measurement of borneol, isoborneol, and camphor

A Trace GC Ultra Gas Chromatograph (Thermo Fisher Scientific, Milan, Italy) was coupled to a Trace DSQ II single quadrupole mass spectrometer (Thermo Fisher Scientific, Austin, TX, USA). Aliquots (20 μL) of n-hexane extract were introduced into a PTV injector with a glass sintered deactivated liner (Part No 45322060; ∼1.2 mm ID, 2.75 mm OD, 120 mm length, 135 μL theoretical volume) in the LVI mode at an injection temperature of 60 °C, an injection time of 0.2 min, a helium gas flow rate of 20 mL/min, a transfer heating rate of 14.5 °C/s, a transfer temperature of 280 °C, and a splitless time of 1 min. Subsequently, the cleaning temperature was increased to and maintained at 320 °C for 8 min at a helium gas flow rate of 300 mL/min to clean any remaining impurities. The chromatographic separation was achieved on a TR-5MS capillary column (30 m×0.25 mm ID; Thermo Fisher Scientific, Cheshire, UK) coated with 5% phenyl polysilphenylene-siloxane (0.25 μm film thickness), and the helium carrier gas (99.999% purity) was delivered at a rate of 1.5 mL/min. The following GC temperature program was used: 60 °C (0–2 min), 60 °C to 132 °C (2–11 min at 8 °C/min), 132 °C to 240 °C (11–12 min at 108 °C/min), 240 °C (12–14 min), and 240 °C to 60 °C (14–17 min at −60 °C/min). The mass spectrometry was operated in electron impact (EI) mode at 70 eV. The temperatures set for the transfer line and the ion source were identical, at 250 °C. Selected ion monitoring of borneol, isoborneol, camphor, and the internal standard naphthalene was achieved at m/z 95, 95, 95, and 128, respectively. Matrix-matched calibration curves were constructed for borneol, isoborneol, and camphor (0.25–16 000 nmol/L for each analyte) using weighted (1/X) linear regression of the analyte/internal standard peak area ratios (Y) against the corresponding nominal plasma concentrations of the analytes (X, nmol/L). The plasma concentration of the internal standard was 125 nmol/L.

Liquid-liquid microextraction for plasma sample clean-up

Thawed plasma samples (60 μL) were spiked with 10 μL of the internal standard solution and then extracted with 35 μL of n-hexane in glass tubes by vortex mixing at 1600 r/min for 3 min. After a short centrifugation, the supernatant organic extracts were aspirated into 100 μL glass inserts of a small internal diameter (placed in 1.5 mL polypropylene microcentrifuge tubes) and centrifuged at 21 000×g for 10 min. After centrifugation, the inserts were carefully placed in 2 mL sampler vials, and aliquots (20 μL) of the supernatants were used for GC-MS analysis.

Determination of analyte solubility

Borneol, isoborneol, camphor, and the internal standard naphthalene, as well as a variety of nonvolatile compounds (quercetin, ginkgolide A, orcinol glucoside, tanshinol, and cinnamic acid), were assessed for their solubility in phosphate buffered saline (PBS; pH 7.4) at 23 °C. In brief, a volume of 100 μL of PBS was added into a 10 mL glass centrifuge tube containing approximately 50 mg of solid test compound, and then the tube was sealed with parafilm. The suspension was sonicated for 10 min and vortexed at 1600 r/min for 5 min, which was repeated twice to enhance compound dissolution. After cooling to 23 °C, a small amount of the solid compound was added into the tube to promote precipitation. The resealed tube was kept under constant agitation and sampled at 24, 32, and 48 h. After centrifugation at 21 000×g for 10 min, the supernatant was diluted and measured by either GC–MS (for the volatile compounds) or LC-MS/MS (for the nonvolatile compounds). The compound solubility was established when equilibrium was achieved as determined by the constant concentrations measured with the two serial samples. A similar study was performed for the test compounds to assess their solubility in either n-hexane or ethyl acetate, except for tanshinol and cinnamic acid. The solubility of tanshinol in ethyl acetate was assessed by extracting the saturated aqueous solution of sodiated tanshinol with an equal volume of the organic solvent. The same procedure was performed to measure the solubility of cinnamic acid in ethyl acetate.

Assessment of the effects of the organic solvent-plasma volume ratio on the extraction efficiency and analyte response

Eighty microliters of rat plasma fortified with 200 nmol/L of borneol, isoborneol, camphor, or naphthalene were extracted in a glass tube with 20, 40, 80, 160, 320, or 640 μL of n-hexane to produce solvent-plasma volume ratios of 0.25, 0.5, 1, 2, 4, and 8, respectively. After vortex mixing and centrifugation, the n-hexane extract supernatant (2 μL) was used for GC-MS analysis. The extraction efficiency was determined by division of the analyte response (peak area) of the n-hexane extract versus that of the control n-hexane solution at the same compound concentration. A similar procedure was also performed using ethyl acetate as the extracting solvent for these volatile compounds, as well as the nonvolatile compounds ginkgolide A, quercetin, orcinol glucoside, tanshinol, and cinnamic acid. In addition, tanshinol and cinnamic acid were further tested individually using plasma samples acidified with 1 mol/L hydrochloric acid to adjust the pH to 1.2.

Assessment of matrix effects on GC-MS-based assay

The potential acute matrix-induced response enhancement34,35 was assessed using a post-extraction spike method36,37 for the PTV-based LVI involved in the GC-MS measurement of plasma levels of borneol, isoborneol, camphor, and the internal standard naphthalene. The matrix effects on the measurement of the analytes in plasma were examined by comparing the peak area of the test compounds between two different sample sets. In sample set 1, the test compounds were dissolved in matrix-free n-hexane and analyzed at concentrations of 3.91, 250, and 16000 nmol/L, except for the internal standard at 125 nmol/L. In sample set 2, blank rat plasma samples obtained from five rats were extracted with a half volume of n-hexane, and the resulting extracts were spiked with the analytes (3.91, 250, or 16000 nmol/L) and the internal standard (125 nmol/L). Division of the mean peak area of sample set 2 versus sample set 1 would indicate the possibility of an enhanced response (absolute matrix effect). When the absolute matrix effect was greater than 120%, additional variability of the peak area in sample set 2 versus that in sample set 1 would indicate the presence of a relative matrix effect.

A long-term study was implemented to assess the tolerance of the PTV injector and the GC capillary column with 100 successive injections (20 μL for each injection) of plasma extract standard containing borneol, isoborneol, camphor, and the internal standard naphthalene (250 nmol/L for each compound). The potential subchronic matrix effect was determined with regards to changes of the compound response (peak area), the column efficiency, the retention time, and the peak shape.

GC-MS-based assay validation

Assay validation was carried out according to the US FDA guidelines on bioanalytical method validation (http://www.fda.gov/cder/guidance/index.htm) to demonstrate that the newly developed bioanalytical method was reliable for its intended applications. The quality control samples were prepared from an independent weighing of the reference standards.

Application of assay to pharmacokinetic studies and blood sampling in rats

Male Sprague-Dawley rats (∼250 g) were obtained from the Sino-British SIPPR/BK Animal Laboratory (Shanghai, China). The use of rats and study protocols was approved by the IACUC (Shanghai, China). For blood sampling, a polyurethane cannula was cannulated aseptically into the femoral artery of each rat under pentobarbital anesthesia. After regaining their preoperative weights, the femoral artery-cannulated rats were randomly assigned to five groups (three rats/group) and received a single oral dose of Bingpian at 20 mg borneol/kg, synthetic Bingpian at 20 mg borneol+isoborneol/kg, or Fufang-Danshen tablet preparation at 20 mg borneol+isoborneol/kg or a single intravenous dose of Bingpian at 5 mg borneol/kg or synthetic Bingpian at 5 mg borneol+isoborneol/kg. For the oral administration, Bingpian, synthetic Bingpian, and Fufang-Danshen tablets were prepared separately by grounding and mixing them with 5% (w/v) Tween-80 to achieve concentrations of 1 mg borneol/mL, 1 mg borneol+isoborneol/mL, and 1 mg borneol+isoborneol/mL, respectively. For the intravenous administration, grounded Bingpian and synthetic Bingpian were dissolved in a mixture containing 0.5% (w/v) N,N-dimethylacetamide, 9.5% (w/v) cremophor EL, and 90% (v/v) saline to achieve a concentration of 1 mg borneol/mL and 1 mg borneol+isoborneol/mL, respectively. Serial blood samples (130–150 μL; 0, 5, 15, 30 min, and 1, 2, 4, 6, 8, 10, 14, and 24 h) were collected into heparinized tubes and centrifuged. The resulting plasma fractions were kept frozen at −70 °C until analysis. The plasma PK parameters were estimated using the non-compartmental model of the Kinetica 5.0 software (Thermo Fisher Scientific, Philadelphia, PA, USA).

Results

GC-MS-based measurement of borneol, isoborneol, and camphor and PTV-based LVI enhances assay sensitivity

In the EI-mass spectra (Figure 2), the molecular ions of borneol, isoborneol, and camphor (m/z 154, 154, and 152, respectively) are present at a low tendency. Accordingly, the most abundant fragment ions at m/z 95 were chosen for the sensitive measurement of these compounds. In contrast, the most intense ion of the internal standard naphthalene was its molecular ion at m/z 128 rather than the fragment ions. To facilitate refocusing of all the measured compounds at the top of the capillary column, the initial temperature of the column oven was set to 60 °C, which was 9 °C lower than the bp of n-hexane. This temperature was maintained for 2 min for complete splitless transfer and column equilibrium. As shown in Figure 2, the chromatographic peaks of borneol (tR, 9.22 min), isoborneol (9.05 min), camphor (8.75 min), and naphthalene (9.57 min) were completely separated from one another and shaped evenly under an optimal column oven heating rate of 8 °C/min.

Figure 2
figure 2

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

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To further increase assay sensitivity, PTV-based LVI (15–30 μL; using the ∼135-μL glass sintered deactivated liner) was optimized and compared with PTV-based splitless injection (2 μL) and PTV-based delayed splitless injection (5–15 μL). The important solvent split parameters for the PTV-based LVI were the injection temperature, the solvent split time, and the solvent split flow-rate; optimization was achieved on the basis of the separation of the solvent n-hexane from the most volatile compound camphor. In addition, the transfer heating rate, transfer temperature, and initial GC column temperature were also important because efficient transfer of the test compounds from the liner to the column was required after solvent elimination. As shown in Figure 3, the best result was obtained with the PTV-based LVI of 20 μL of a standard n-hexane solution containing borneol, isoborneol, camphor, and naphthalene. Increasing the injection size to 30 μL resulted in increased responses, but with considerable effects on the chromatographic peak shapes and vice versa. The unsuccessful implementation of PTV-based LVI over a volume of 20 μL was due to the insufficiently large differences in the bp between the measured compounds and the solvent n-hexane. However, a larger injection size (up to 50 μL) was successfully achieved when the more volatile solvent n-pentane (bp 36 °C) was used instead of n-hexane (69 °C). Although n-pentane was a more favorable solvent for PTV-based LVI, it was rejected for the sample preparation (due to excessively low viscosity) and for the GC-MS analysis with a long waiting time (due to excessively high volatility).

Figure 3
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.

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Liquid-liquid microextraction enhances assay sensitivity

Although n-hexane is an efficient solvent for extraction of borneol, isoborneol, camphor, and the internal standard naphthalene, evaporative losses of these volatile compounds when extracting from plasma occur upon the subsequent dry-down step. For traditional liquid-liquid extraction, the extracting solvent is often used in a solvent-to-sample volume ratio ranging from 3 to 10. The use of a large volume of extracting solvent is supposed to enhance the extraction efficiency. However, excessive solvent levels in this extraction method have a considerable dilution effect, i.e., the analyte concentration in the organic extract is lower than that in the pre-extracted aqueous sample. The application of such an organic extract (without any evaporative reduction) to GC-based analysis will result in decreased assay sensitivity. In the current study, we investigated the effect of the organic solvent-to-plasma volume ratio on the liquid-liquid extraction efficiency. As shown in Figure 4A, the extraction recoveries of borneol, isoborneol, camphor, and naphthalene from plasma using n-hexane as the solvent were almost constant with the decreasing n-hexane-to-plasma volume ratio. This consistency enabled the use of a small volume of extracting solvent for sample preparation to enhance the assay sensitivity by counteracting the aforementioned dilution effect, which was referred to as “liquid-liquid microextraction”. For ease of operation, a small n-hexane-to-plasma volume ratio of 0.5 was chosen for preparation of the plasma samples (70 μL) to ensure an enriched level of test compounds in the organic phase. Aside from the enhancement of assay sensitivity, this newly developed liquid-liquid microextraction method had other advantages, including organic solvent economy, environmental friendliness, and improved assay throughput.

Figure 4
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 elution41. 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.

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To extend the application of the liquid-liquid microextraction method, ethyl acetate was also tested as a solvent for recovering borneol, isoborneol, camphor, and naphthalene from plasma. The results suggested that the performance of ethyl acetate for the compound extraction was similar to that of n-hexane. Although the relatively high bp (77 °C) is unfavorable for the PTV-based LVI used in the current study, ethyl acetate is commonly used in sample preparation for many nonvolatile compounds. Figure 4B shows the results of the ethyl acetate-based liquid-liquid extraction of several nonvolatile medicinal herbal compounds from plasma. The extraction efficiency was high for quercetin and ginkgolide A in an ethyl acetate-to-plasma volume ratio-independent manner. However, the extraction of orchinol glucoside, tanshinol, and cinnamic acid was significantly dependent on the ethyl acetate volume used; thus a large volume of the solvent was needed for high extraction recovery. The observed inconsistency in extraction performance appeared to be related to the organic solvent-to-water solubility ratios (SO/SW ratios) of the test compounds (Table 1). When the pH value of the aqueous phase was 7.4, the measured solubility ratios of borneol, isoborneol, camphor, naphthalene, quercetin, and ginkgolide A were high (122–9435; using n-hexane or ethyl acetate as the organic solvent), but those of orchinol glucoside, tanshinol, and cinnamic acid were very low (0.0006–0.137; using ethyl acetate as the organic solvent). These results could be predicted because the calculated logD values (pH 7) of the compounds were well correlated with their measured solubility ratios (Table 1). The logD values (pH 7) of borneol, isoborneol, camphor, naphthalene, quercetin, and ginkgolide A ranged from −0.14 to 3.36, which were greater than those of orchinol glucoside, tanshinol, and cinnamic acid (−4.73 to −0.28), with a logD value of >–0.2 serving as the threshold. Tanshinol and cinnamic acid are weak acids with low ionization constants (pKa) of 3.3 and 4.5, respectively. Liquid-liquid extraction efficiency is significantly affected by the existing state of the test compound in the aqueous phase because the compound in either its ionized form or molecular form favors the aqueous phase or the organic phase, respectively. Accordingly, plasma samples that were spiked with either tanshinol or cinnamic acid were acidified with 1 mol/L hydrochloric acid to a pH of 1.2. The acidification resulted in increased ethyl acetate-based extraction efficiency for cinnamic acid but not for tanshinol (Figure 4C). As shown in Table 1, the effect of acidification on the enhanced extraction efficiency appeared to be predictable by comparing the logD values (pH 7 and pH 1) of the compounds. The logD values (pH 7 and pH 1) of cinnamic acid were −0.38 to −1.77 and 1.21 to 2.16, respectively, whereas those of tanshinol were −3.77 to −4.73 and −0.28 to −1.15, respectively.

Table 1 Physicochemical properties of borneol, isoborneol, camphor, and naphthalene, as well as those of quercetin, ginkgolide A, orcinol glucoside, tanshinol, and cinnamic acid.
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Assay reliability and sensitivity

The potential acute matrix-induced response enhancement was assessed during assay development. As a result, the matrix components from the n-hexane-extracted plasma samples had a negligible influence on the measurement of borneol, isoborneol, camphor, and the internal standard naphthalene, demonstrating absolute matrix effects of 91.5%–104%, 93.3%–109%, 89.6%–107%, and 103%, respectively. Accordingly, calculation of the relative matrix effects was unnecessary. As shown in Table 2, the compound responses and the chromatographic behaviors (eg, column efficiency, retention times, and peak shapes) did not change significantly during and after 100 successive injections of the plasma extract standard, indicating good tolerance of the PTV injector and the GC column for long-term use with biomatrix samples.

Table 2 Long-term stability of the measurement of the plasma extract standard containing borneol, isoborneol, camphor, and the internal standard naphthalene (250 nmol/L for each compound). The data are expressed as the mean±standard deviation (relative standard deviation).
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The analytes borneol, isoborneol, camphor, and the internal standard naphthalene were stable under analytical conditions mimicking those experienced by real plasma samples during an assay run. The calibration curves for the measurement of the analytes exhibited good linear ranges from 0.98 to 16000 nmol/L with correlation coefficients >0.99. As shown in Table 3, the within-run (n=5) and between-run (n=3) assay precision, assessed at the nominal plasma concentrations 0.98, 250, and 16 000 nmol/L, were reliable (4.5%–9.6% for borneol, 5.9%–9.0% for isoborneol, and 3.1%–7.2% for camphor), and the associated assay accuracies were also satisfactory (90.0%–111% for borneol, 94.0%–109% for isoborneol, and 98.5%–113% for camphor). Accordingly, the lower quantification limit of 0.98 nmol/L was acceptable for the measurement of borneol, isoborneol, and camphor in plasma. To the best of our knowledge, the sensitivity of our newly developed assay is notably higher than that reported for the measurement of the monoterpene alcohols in plasma (32.5–325 nmol/L)27,28,29,30,31,32,33.

Table 3 Assay accuracy and precision (relative standard deviation) for the measurement of borneol, isoborneol, and camphor in rat plasma. The data are expressed as the mean±standard deviation (relative standard deviation).
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Application of the novel assay in the pharmacokinetic study of Bingpian, synthetic Bingpian, and Fufang-Danshen tablets

The pharmacokinetic study of Bingpian, synthetic Bingpian, and the synthetic Bingpian-containing phytopharmaceutical product Fufang-Danshen tablets in rats is an ideal application of the newly developed assay. Figure 5 depicts the plasma level-time profiles of borneol, isoborneol, and their metabolite camphor; the associated plasma pharmacokinetic parameters are summarized in Table 4. The oral bioavailability (F) of borneol was low (8.3%–14.5% for Bingpian, 11.9%–14.0% for synthetic Bingpian, and 10.9%–17.8% for the Fufang-Danshen tablets). The F values of isoborneol were also low (2.0%–4.9% for synthetic Bingpian and 4.8%–12.7% for the Fufang-Danshen tablets). The metabolism from borneol and isoborneol to camphor appeared to contribute to the first-pass elimination of the monoterpene alcohols. This was indicative of the mean ratio of “camphor”-to-“borneol+isoborneol” in AUC0–∞ (0.72) after oral administration of synthetic Bingpian was greater than the ratio (0.29) after intravenous administration of the same drug. The mean ratio of “camphor”-to-“borneol” after oral ingestion of Bingpian was approximately 4 times greater than the ratio after intravenous administration of Bingpian. The plasma concentrations of borneol after the intravenous dose of either Bingpian or synthetic Bingpian declined in a biphasic fashion with first mean half-lives (t1/2–(1)) of 0.7–0.8 h and second mean half-lives (t1/2–(2)) of 8.0–8.5 h, and the plasma concentrations of isoborneol declined rapidly (t1/2–(1), 1.0–1.3 h). The mean total plasma clearance (CLtot,p) values of borneol were 12.3 L·h−1·kg−1 (for Bingpian) and 8.6 L·h−1·kg−1 (for synthetic Bingpian) and the mean CLtot,p of isoborneol was 13.7 L·h−1·kg−1 (for synthetic Bingpian). The mean distribution volumes at steady state (VSS) of borneol (40.2 and 50.7 L/kg for Bingpian and synthetic Bingpian, respectively) and isoborneol (13.0 L/kg for synthetic Bingpian) exceeded the reported volume of rat total body water (0.67 L/kg)38, suggesting that borneol and isoborneol tended to bind to the tissue components. Borneol was not transformed to isoborneol in rats, which was indicated by the lack of isoborneol detected in plasma after the administration of Bingpian.

Figure 5
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).

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Table 4 Plasma pharmacokinetics of borneol, isoborneol, and camphor in rats receiving Bingpian, synthetic Bingpian, or Fufang-Danshen tablets. The data are expressed as the mean±standard deviation (relative standard deviation).
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Discussion

The use of herbal therapies is escalating worldwide. In correlating the administration of herbal medicines with their beneficial effects, it is important to understand the absorption, disposition, and pharmacokinetics of their active ingredients after dosing. In addition to nonvolatile ingredients, many volatile constituents are also pharmacologically important for herbal therapies. However, the assays available for measuring volatile compounds have not been very successful in supporting the associated pharmacokinetic studies. Herein, we developed a reliable assay for the sensitive measurement of borneol, isoborneol, and their metabolite camphor in plasma to support a pharmacokinetic study of Bingpian. The high sensitivity of this assay was achieved by using a liquid-liquid microextraction technique in concert with PTV-based LVI of the organic extract for GC-MS, which was 33–330 times higher than the sensitivity of earlier reported analytical methods for Bingpian27,28,29,30,31,32,33. To our knowledge, this is the first time that liquid-liquid microextraction has been combined with PTV-based LVI and developed to measure the concentrations of volatile herbal compounds in complex biomatrices. Our study provides a simple, effective solution to the analytical problem hampering the implementation of pharmacokinetic studies of herbal medicines containing volatile ingredients of pharmacological importance.

Several liquid-liquid microextraction techniques such as single-drop microextraction, hollow fiber liquid phase microextraction, and dispersive liquid-liquid microextraction have been developed39. However, these techniques have some disadvantages, including tedious and labor-consuming operation, the requirement of special equipment, and applicability to a limited range of analytes. Recently, Serrano et al developed a similar liquid-liquid microextraction combined with PTV-based LVI for water analysis to measure haloacetaldehyde concentrations in swimming pool water40. In this current study, the developed method of liquid-liquid microextraction combined with PTV-based LVI is quite feasible and can be routinely used for pharmacokinetic studies of Bingpian and the associated herbal products. The utilization ratio of the injection volume (20 μL) to the extracting solvent volume (35 μL) was high (57%). Our work is an important addition to previous studies associated with miniaturized liquid-liquid extraction techniques because we have addressed the application of this technique in the analysis of volatile and nonvolatile compounds, which could be predicted according to the analytes' logD and pKa. Compounds with logD>−0.2 appeared to be more suitable for such liquid-liquid microextraction.

Acute matrix-induced response enhancement has been observed for GC-based analyses of pesticide residues in fatty foods34,35. The matrix protects the analytes from both adsorption and alterations during transfer from the injector (especially in the injection liner) to the capillary column. The matrix effect results in the response of the analyte from a matrix sample being higher than that from a matrix-free solution containing the compound at the same concentration. In this study, the acute matrix-induced response enhancement was almost negligible for the measurement of plasma concentrations of borneol, isoborneol, camphor, and the internal standard naphthalene. In addition, the compound responses and the chromatographic behaviors did not change significantly during and after 100 successive injections of the plasma extract standard, indicating good tolerance of the PTV injector and the GC column for long-term use with plasma samples. It has been reported that polar compounds (containing phosphate, hydroxyl, amino, imidazole, benzimidazole, carbamate, and urea functional groups), as well as strong acids and/or bases, are more susceptible to matrix-induced enhancement35. Borneol, isoborneol, camphor, and naphthalene are nonpolar and neutral compounds, which might explain the observed negligible matrix effects on the GC-MS-based measurement of the analytes.

In summary, a reliable GC-MS-based assay for the sensitive measurement of plasma concentrations of borneol, isoborneol, and their metabolite camphor was developed, primarily by using liquid-liquid microextraction in concert with PTV-based LVI of the organic extract. The application of the miniaturized liquid-liquid extraction technique could be extended to the measurement of other volatile compounds and many nonvolatile medicinal compounds in biomatrices, which can be predicted according to the logD and pKa of the analytes. On the basis of this newly developed assay, it was observed that, in rats, isoborneol was more likely to be metabolized into camphor than borneol.

Author contribution

Chuan LI, Chen CHENG, and Yan SUN designed the research; Chen CHENG, Xin-wei LIU, Fei-fei DU, Mei-juan LI, Fang XU, Feng-qing WANG, and Yang LIU, performed the research; Chuan LI, Chen CHENG, Xin-wei LIU, and Yan SUN analyzed data; Chuan LI and Chen CHENG wrote the paper.