Abstract
A very simple and selective high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC–MS-MS) method was developed for simultaneous determination and pharmacokinetic study of protocatechuic aldehyde (PAL) and its active metabolite protocatechuic acid (PCA). The method involves a simple liquid–liquid extraction with ethyl acetate. The separation was performed on a Hypersil GOLD C18 column (2.1 × 150 mm, 3.0 µm; particle, Thermo, USA) with isocratic elution using a mobile phase consisted of methanol and water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min. The detection of target compounds was done by using low-energy collision dissociation tandem mass spectrometry (CID–MS-MS) using the selective reaction monitoring scan mode. The method was linear for all analytes over the investigated range for all correlation coefficients greater than 0.9950. The lower limits of quantification were 2.0 ng/mL for PAL and PCA. The intra- and interday precisions (relative standard deviation, RSD %) were <6.84 and 5.54%, and the accuracy (relative error, RE %) was between −2.85 and 0.74% (n = 6). The developed method was applied to study the pharmacokinetics of PAL and its major active metabolite PCA in rat plasma after oral and intravenous administration of PAL.
Introduction
The dried root of Salvia miltiorrhiza (Danshen), a commonly used traditional Chinese medicine (TCM), is widely used to treat coronary heart disease, cerebrovascular disease, hepatitis, hepatocirrhosis and chronic renal failure (1–4). It is mainly distributed throughout the north of China and is officially listed in the Chinese Pharmacopoeia (5). There are a number of traditional Chinese medicinal preparations (TCMPs) that contain Danshen. Protocatechuic aldehyde (PAL) has been considered to be one of the major active constituents of S. miltiorrhiza. A number of pharmacological studies have shown that PAL possessed biological activities, such as improving microcirculation, reducing atherosclerosis, inhibiting the aggregation of platelets and anti-oxidant and anti-inflammatory effects (6–10). Protocatechuic acid (PCA; 3,4-dihydroxybenzoic acid) is a water-soluble monomeric phenolic acid compound, which is widely distributed in the nature of various kinds of medicinal plants. PCA plays an important role in anti-radical, anti-oxidant, anti-bacterial and anti-tumor promotion activities (11–13). PCA is a major bioactive metabolite of PAL in the previous study (14). Metabolite identification is important in the drug discovery and development process to optimize lead compounds for further development. Now the study of metabolites' exposure levels attracts more and more attention. We must pay attention to the role of active metabolites in the study of new drugs, because sometimes the pharmacological activities of metabolites relate to the safety and efficacy. The FDA provides guideline about the drug systemic exposure of the metabolites (15).
Many methods, including high-performance liquid chromatography (HPLC) with UV detection, ultra high-performance liquid chromatography (UHPLC), HPLC coupled to mass spectrometry (LC–MS) or tandem mass spectrometry (LC–MS-MS), have almost completely replaced other detection systems in the field of bio-analytical research because of their high sensitivity and specificity. Furthermore, tandem mass spectrometry techniques have been playing an important role in the pharmacokinetic study, especially in simultaneous determination of prototype drugs and their metabolites (16–19). There are some reports concerning the determination of PAL and PCA by HPLC (20–22), LC–MS (23) and LC–MS-MS (24) methods. However, no method is reported for simultaneous quantification of PAL and PCA in plasma. Therefore, it is necessary to develop a sensitive and reliable method for simultaneous quantification of PAL and PCA.
Our study presents a simple LC–MS-MS method for the simultaneous quantification of PAL and its active metabolite PCA in rat plasma. This method was successfully applied for the evaluation of pharmacokinetic profiles of PAL and its active metabolite PCA in rats after oral and intravenous administration of PAL.
Experimental
Materials and reagents
The reference standards of PAL (>98% purity), PCA (>98% purity) and Danshensu (DSS, >98% purity, internal standard, IS, structure as shown in Fig. 1) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Methanol (HPLC grade) was purchased from Merck (Darmstadt, Germany). Formic acid of HPLC grade was obtained from Tedia Company Inc. (Beijing, China). Ultrapure water was prepared from a Milli-Q Reagent system (Millipore, Billerica, USA). Other reagents were of analytical grade.
Figure 1.
Chemical structures and product ion mass spectra of A (PAL); B (PCA) and C (IS; DSS). This figure is available in black and white in print and in color at JCS online.
Instrumentation and chromatographic conditions
The HPLC system consisted of a Surveyor pump (Thermo Finnigan, USA) and a Surveyor auto-sampler (Thermo Finnigan, USA). Chromatographic separation was performed by using a Hypersil GOLD C18 (2.1 × 150 mm, 3.0 µm, particle, Thermo, USA) analytical column protected by a Hypersil GOLDDrop-in guard column (3 µm, particle, Thermo, USA) at 30°C. The mobile phase consisted of (A) aqueous formic acid (0.1%, v/v) and (B) methanol with isocratic elution (A:B; 77:23; v/v) at a flow rate of 0.2 mL/min. The analytical time was 7.0 min for each injection. Mass spectrometric detection was performed on a Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source (San Jose, CA, USA). Data processing was carried out on Xcalibur® (version 1.3) software. The mass spectrometer was operated in the negative mode. Quantification was obtained using select reaction monitoring (SRM) at m/z transitions of m/z 137 → 107.8 for PAL, m/z 153 → 109 for PCA and m/z 197 → 135 for IS, respectively (Fig. 1). The MS parameters are as follows: spray voltage: 3.0 kV; heated capillary temperature: 350°C; sheath gas (nitrogen): 20 psi; the auxiliary gas (nitrogen): 5 psi; and the collision gas (argon) pressure: 1.5 m Torr. The collision energy was as follows: 23 eV for PAL; 15 eV for PCA; and 19 eV for IS. MS–MS operating conditions were optimized by infusion of the standard solution (1 µg/mL) of each analyte and IS into the ESI source via a syringe pump.
Preparation of standard solutions
The standard stock solutions of PAL, PCA and the IS were prepared in methanol at the concentration of 1 mg/mL. The working standards were prepared by dilution of the stock solution in methanol to obtain the desired concentrations. All standard solutions were stored at 4°C for further use.
Sample preparation
All frozen standards and samples were allowed to thaw at room temperature. The liquid–liquid extraction procedure was utilized for the sample preparation. To a 100 µL aliquot of the rat plasma sample, IS (100 µL, 500 ng/mL) and 100 µL of 1 mol/L hydrochloric acid solution were added. After vortexing for 30 s, a 3 mL of the ethyl acetate was added to the sample. The mixture was vortex-mixed for 3 min and then centrifuged for 10 min at 4,500 rpm. The upper organic layer was removed and evaporated to dryness at 30°C under a stream of nitrogen. The residues were reconstituted in 100 µL methanol—water (50:50, v/v). A 20 µL of the solution was injected into the LC–MS-MS system for analysis.
Method validation
Specificity and selectivity
The specificity of the method was evaluated by analyzing six different sources of blank plasma samples. By comparing the SRM chromatograms of the blank plasma samples with those of the corresponding spiked plasma samples at the lower limits of quantification (LLOQ) level, the response of coeluting interferences should be <20% of the analytes and <5% of the peak area of IS.
Linearity and LLOQ
The calibration curves were prepared by assaying standard plasma samples at seven concentration levels. The linearity of each calibration curve was determined by plotting the peak area ratio (y) of analytes to IS versus the nominal concentration (x) of analytes with weighted (1/χ2) least square linear regression. The LLOQ for the analytes was the lowest concentrations with a signal-to-noise of ≥ 5, which could be quantitatively determined with precision and accuracy ≥20%, evaluated by analyzing samples in six replicates.
Precision and accuracy
The precision and accuracy were assessed by analyzing quality control (QC) samples in six replicates at low, medium and high QC of the analytes in six replicates on the same day and on three different days, respectively. The precision was defined as relative standard deviation (RSD) of the measured concentration, and the accuracy as the relative error (RE) of the measured mean value deviated from the nominal value.
Extraction recovery and matrix effect
The extraction efficiency of the analytes and IS was determined by analyzing six replicates of plasma samples at low, medium and high QC levels. The recovery was evaluated by comparing the peak area of extracted plasma standard with the peak area of post-extraction plasma blank spiked with equivalent concentrations using six replicates. The matrix effect (ME) was defined as the ion suppression/enhancement on the ionization of analytes, which was evaluated by comparing the responses of the deproteinized sample of blank plasma from six spiked QC samples with those of the standard samples at equivalent concentrations.
Stability
The stability of each analyte in rat plasma was assessed by analyzing three concentration levels of low, medium and high QC samples at different conditions. The post-preparation stability was tested by determining the extracted QC samples stored in the auto-sampler (4°C) for 24 h. The freeze and thaw stability was determined after three freeze–thaw cycles (−20°C). Long-term stability in rat plasma stored at −20°C was studied for a period of 1 month employing QC samples at three levels.
Application of the method in a pharmacokinetic study
Male Wistar rats, weighing (240 ± 20) g, were provided by Vital River Lab Animal Technology Co., Ltd (Beijing, China) and housed with a 12 h light/12 h night cycle at an ambient temperature (about 25°C) and 60% relative humidity. Free access to food and water were allowed at all times except for fasting 12 h before the experiment. All animal experiments were carried out according to the Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Tianjin Tasly Institute. After fasting for 12 h prior to the experiment, 12 rats were randomly assigned to two groups, and six rats were given PAL at a single dose by oral (20 mg/kg) and intravenous (1 mg/kg) administration through tail vein, respectively. Blood samples (0.25 mL) were obtained from the suborbital vein at 0 (pre-drug), 0.03, 0.08, 0.17, 0.33, 0.67, 1, 2, 4 and 6 h after intravenous administration and 0 (pre-drug), 0.08, 0.17, 0.25, 0.50, 0.75, 1, 2, 4, 6 and 8 h after oral administration. The plasma was separated by centrifuging the blood at 4,500 rpm for 10 min and was stored frozen at −20°C until analysis.
Results
Optimization of chromatography-mass spectrometric conditions
In this experiment, the chromatographic conditions were optimized to improve the peak shape, increase the signal response of analytes and shorten the run time. The best peak shape and ionization were achieved by using 0.1% formic acid. Finally, the mobile phase that consisted of (A) aqueous formic acid (0.1%, v/v) and (B) methanol with isocratic elution (77:23, v/v) at a flow rate of 0.2 mL/min was used.
The intensity response of the ion signals observed was higher when using the negative ionization mode than the positive mode. In the precursor ion scan spectra, the most abundant ions were protonated molecules [M–H]− m/z 137, 153 and 197 for PAL, PCA, and for IS, respectively. By manual optimization using infusion with a syringe pump, the most suitable heated capillary temperature, spray voltage and collision energy with SRM for all target analytes were selected. As shown in Figure 1, those precursor and product ions were m/z 137 → 107.8 for PAL, m/z 153 → 109 for PCA and m/z 197 → 135 for IS, respectively.
Method validation
Specificity and selectivity
The typical chromatograms obtained from a blank, a spiked plasma sample with the analytes (at LLOQS) and IS and a plasma sample after an oral administration of PAL are shown in Figure 2. No obvious interferences from endogenous plasma substances were observed under the chromatographic conditions.
Figure 2.
Representative SRM chromatograms of the analytes components and IS in plasma: (A) blank plasma sample, (B) blank plasma spiked with the components in LLOQ, (c) real plasma sample obtain 2 h after oral administration of PAL (20 mg/kg). Peak 1, PAL; Peak 2, PCA; Peak 3, IS. This figure is available in black and white in print and in color at JCS online.
Linearity and LLOQ
The calibration curves were obtained by plotting the peak area ratio of the analytes to IS against the corresponding concentration of the analytes in the prepared plasma calibrators. The plasma calibration curves of PAL and PCA were constructed using seven calibration standards (2.0–5,000 ng/mL). The linear regression equation of the curve for PAL was y= 0.0211x − 0.0001 (r= 0.9950) and that for PCA was y= 0.0198x +0.0101 (r= 0.9959). They all exhibited good linearity. The LLOQs of PAL and PCA were 2.0 ng/mL. The LLOQ was sufficient for pharmacokinetic studies after oral and intravenous administration of PAL to rats.
Precision and accuracy
The assay precision and accuracy results are shown in Table I. The intra- and interday precisions were ranged from 1.08 to 8.31% and 1.11 to 12.50%, respectively, and the accuracy was ranged from −3.75 to 0.74%. The results indicated that the values were all in the acceptable ranges.
Table I.
Precision, Accuracy and Extraction Recoveries of Analytes
| Analytes | Spiked (ng/mL) | Intraday (n = 6) |
Interday (n = 18) |
||||
|---|---|---|---|---|---|---|---|
| Concentration measured (ng/mL) | Precision (%, RSD) | Accuracy (%, RE) | Concentration measured (ng/mL) | Precision (%, RSD) | Accuracy (%, RE) | ||
| PAL | 2 | 1.94 ± 0.12 | 7.17 | −3.00 | 2.02 ± 0.16 | 11.12 | 1.13 |
| 5 | 4.93 ± 0.29 | 5.93 | −1.36 | 5.02 ± 0.25 | 5.02 | 0.30 | |
| 100 | 97.14 ± 4.88 | 5.03 | −2.85 | 99.18 ± 4.96 | 4.99 | −0.81 | |
| 4,000 | 4,023.50 ± 43.55 | 1.08 | 0.59 | 4,001.22 ± 48.98 | 1.22 | 0.33 | |
| PCA | 2 | 1.93 ± 0.11 | 8.31 | −3.75 | 1.98 ± 0.19 | 12.50 | −0.70 |
| 5 | 4.96 ± 0.34 | 6.84 | −0.7 | 5.04 ± 0.28 | 5.54 | 0.74 | |
| 100 | 100.31 ± 5.72 | 5.70 | 0.32 | 100.07 ± 5.21 | 5.21 | 0.07 | |
| 4,000 | 3,978.16 ± 46.28 | 1.16 | 0.13 | 3,995.66 ± 44.49 | 1.11 | −0.11 | |
Extraction recovery and ME
The extraction recoveries determined for PAL, PCA and IS are shown in Table II. The absolute extraction recoveries of the analytes were all in the range of 70.53–73.54%. The extraction of the IS was 76.02%. These results demonstrated that the values were all in the acceptable ranges. The MEs derived from the QC samples were from 88.77 to 109.54%. These results confirmed that the evaluated method was free from any ME.
Table II.
Extraction Recovery and ME of Analytes and IS
| Analytes Spiked | Concentration (ng/mL) | Extraction recovery (n = 6) |
MEs (n = 6) |
||
|---|---|---|---|---|---|
| Mean ± SD (%) | RSD (%) | Mean ± SD (%) | RSD (%) | ||
| PAL | 5 | 70.53 ± 2.73 | 3.87 | 88.77 ± 1.70 | 1.91 |
| 100 | 72.75 ± 2.35 | 3.23 | 92.59 ± 2.66 | 2.86 | |
| 4,000 | 73.54 ± 0.81 | 1.10 | 97.25 ± 0.63 | 0.64 | |
| PCA | 5 | 73.2 ± 1.71 | 2.33 | 109.54 ± 4.27 | 3.91 |
| 100 | 73.08 ± 1.67 | 2.28 | 106.91 ± 3.11 | 2.91 | |
| 4,000 | 73.54 ± 0.81 | 1.10 | 103.01 ± 2.20 | 2.13 | |
| IS | 500 | 76.02 ± 2.90 | 3.81 | 94.69 ± 3.24 | 3.42 |
Stability experiments
The stability of the analytes during the sample processing procedures and storing was evaluated by analyzing the three levels of the QC samples. The results are shown in Table III. The results of stability tests indicated that the six analytes in plasma were all stable for 1-month storage at −20°C, 24 h in the auto-sampler (4°C) and three freeze–thaw cycles with RSD% in range of 0.48–6.96%.
Table III.
Stability of Analytes
| Analytes | Spiked (ng/mL) | Storage at −20°C for 1 month |
Auto-sampler 4°C for 24 h |
Three thaw cycles |
|||
|---|---|---|---|---|---|---|---|
| Measured concentration | RSD (%) | Measured concentration | RSD (%) | Measured concentration | RSD (%) | ||
| PAL | 5 | 4.98 ± 0.21 | 4.11 | 4.76 ± 0.23 | 4.77 | 5.05 ± 0.32 | 6.24 |
| 100 | 99.84 ± 3.33 | 3.34 | 97.41 ± 6.12 | 6.27 | 97.47 ± 6.79 | 6.96 | |
| 4,000 | 4,011.33 ± 47.25 | 1.17 | 4,004.33 ± 75.64 | 1.89 | 3,933.67 ± 23.06 | 0.58 | |
| PCA | 5 | 5.22 ± 0.26 | 4.93 | 4.98 ± 0.21 | 4.21 | 5.18 ± 0.18 | 3.57 |
| 100 | 98.87 ± 6.36 | 6.44 | 98.15 ± 5.31 | 5.42 | 99.17 ± 3.70 | 3.73 | |
| 4,000 | 4,042.67 ± 19.65 | 0.48 | 3,992.33 ± 30.16 | 0.75 | 3,998.67 ± 63.79 | 1.59 | |
Application to the pharmacokinetic study
The developed method was successfully applied to the pharmacokinetics study of PAL and PCA in rat plasma after intravenous and oral administration of PAL. The mean plasma concentration–time profiles of the analytes in rat plasma are shown in Figure 3. The results indicated that PAL increased rapidly in rats after oral administration and reached the Cmax within 0.25 h. The relatively rapid oral absorption could be due to its low-polarity characteristics and small molecule size. The pharmacokinetic parameters such as the AUC, MRT(0−t), t1/2, tmax and Cmax are listed in Table IV. All pharmacokinetic parameters were processed by a non-compartmental model using DAS 2.0 software. The analytes were rapidly eliminated from rat plasma, and their concentration was below the LLOQ after collection of plasma samples 6 and 8 h after intravenous and oral administration, respectively. The t1/2 for the analytes to rats was 0.66 ± 0.11, 0.50 ± 0.06, 0.92 ± 0.61 and 1.09 ± 0.03 h for intravenous and oral administration, respectively. The relatively short t1/2 indicated that PAL and PCA were distributed and eliminated rapidly in rats. The short t1/2 may result from the fact that the PAL and PCA were easily metabolized in vivo. The level of PAL reached the maximum concentration rapidly and declined promptly. This indicates a spontaneous conversion from PAL to PCA in vivo. As shown in Figure 3, the concentration of PCA was higher than the PAL, and the ratio of AUC0 → ∞ of PCA to PAL was 9.56 ± 2.36 and 6.95 ± 2.24 at intravenous and oral administration, respectively. It may be rapidly converted in vivo like the pro-drug. PAL is extensively metabolized in rats via oxidation (Phase I) pathways. Compared with the prototype drug, the exposure level of the metabolites is very high. The result is remarkable, and we will further research in the future.
Figure 3.
Mean plasma concentration-time profiles of PAL and PCA in rat plasma after intravenous (1 mg/kg) and oral (20 mg/kg) administration of PAL (each point represents mean ± SD, n = 6): A (intravenous); B (oral).
Table IV.
Pharmacokinetic Parameters of PAL and Its Major Metabolite in Rat Plasma After Intravenous and Oral Administration (Mean ± SD, n = 6)
| Parameters | Unit | Intravenous (1 mg/kg) |
Oral (20 mg/kg) |
||
|---|---|---|---|---|---|
| PAL | PCA | PAL | PCA | ||
| t1/2 | h | 0.66 ± 0.11 | 0.50 ± 0.06 | 0.92 ± 0.21 | 1.09 ± 0.03 |
| Cmax | ng/mL | 713.48 ± 240.11 | 2,553.9 ± 572.34 | 706.23 ± 180.73 | 4,860.5 ± 1,794.6 |
| tmax | h | – | 0.80 ± 0.09 | 0.17 ± 0.05 | 0.18 ± 0.06 |
| AUC0−t | ng h/mL | 66.10 ± 9.76 | 653.21 ± 135.52 | 580.43 ± 53.58 | 3,773.9 ± 990.74 |
| AUC0−∞ | ng h/mL | 69.81 ± 9.51 | 657.49 ± 136.62 | 583.47 ± 54.52 | 4,011.1 ± 1558.1 |
| MRT0−t | h | 0.22 ± 0.05 | 0.78 ± 0.17 | 0.66 ± 0.07 | 0.90 ± 0.27 |
| MRT0−∞ | h | 0.34 ± 0.10 | 0.81 ± 0.17 | 0.76 ± 0.11 | 1.32 ± 0.79 |
| AUC0−∞PCA/AUC0−∞ PAL | 9.56 ± 2.36 | 6.95 ± 2.24 | |||
Discussion
The chromatographic separation was performed on a Hypersil GOLD C18 column (150 × 2.1 mm, 3 µm), which contributes to an efficient separation. When the chromatographic conditions were optimized, it was found that methanol resulted in lower background noise and better peak shape. Therefore, methanol was chosen as the organic phase. Furthermore, the use of 0.1% formic acid in the mobile phase improved the peak shape, and the peak tailing was reduced significantly. The selected mobile phase consisted of (A) aqueous formic acid (0.1%, v/v) and (B) methanol with isocratic elution (A:B; 77:23; v/v) and provided little matrix interference and proper retention times. It is necessary to choose a proper IS to achieve good accuracy and precision when a mass spectrometer is adopted in an analysis method. DSS was chosen as the IS because of the similarity of its chemical structure, chromatographic retention time and ionization behavior to that of the analytes.
The aim of this study was to develop and validate a simple and reliable LC–MS-MS method to simultaneous quantification of PAL and PCA in rat plasma. The analysis method was set up by optimizing LC–MS-MS conditions to obtain the best possible sensitivity. The analytes were analyzed first with MS by syringe infusion of individual standard solution, and they were all more efficiently ionized in ESI negative mode than in positive mode. ESI negative mode was therefore employed. SRM was used to monitor the precursor ion and production, which could reduce interference and enhance selectivity. The use of the SRM mode can eliminate the interferences from the rat plasma.
The plasma pretreatment often needed to remove protein and other interferences prior to LC–MS-MS analysis. Ethyl acetate, diethyl ether and trichloromethane were all tested as extraction solvents, and ethyl acetate was finally selected because it could provide high extraction efficiency.
Conclusion
For the first time, a sensitive and selective method for the simultaneous determination of PAL and its active metabolite PCA in rat plasma was developed by using the LC–MS-MS method in negative mode. This developed method was used to perform a pharmacokinetic study in rat plasma after oral and intravenous administration of PAL. This method is specific and sensitive enough to detect lower concentrations of PAL and PCA in rat plasma. The limit of quantification is low enough to monitor at least five half-lives of PAL and PCA concentration with good intra- and interassay reproducibility (% CV) for the quality controls.
Funding
This study was financially supported by the National Key Special Project of Science and Technology for Innovation Drugs of China (Grant No. 2013zx09402202).
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