Application Of Hyphenated Chromatography-Mass Spectrometry Techniques In Analysis Of Plasma Drug Concentration And Constituents Of Traditional Chinese Medicine

Hyphenated chromatography-mass spectrometry techniques combine the chromatographic separation and mass spectrum characterization. With the help of new analytical theory and techniques, the hyphenated techniques provide us a powerful tool and method for fast, simple, selective determination of the target compounds with very low concentration in complex bio-matrix. The two-dimension data produced from the hyphenated instruments provide us much useful information for rapid qualitative and quantitative analysis of a complex mixture system. The development of high-dimensional chemometric resolution approaches can largely enhance our ability of dealing with the complicate analytical system. In our study, using the latest research findings of hyphenated chromatography-mass spectrometry techniques, we developed simple, rapid, selective and accurate analytical methods to detect the target compounds with very low concentration in a complex bio-matrix. The other work in our study is determination of volatile constituents in traditional Chinese medicine. Qualitative and quantitative analysis of the volatile constituents were performed with the help of chemometric methods. In all, contents of this thesis were included as below. 1. Simultaneous determination of five first-line anti-tuberculosis drugs, pyrazinamide (PZA), isoniazid (INH), rifampicin (RFP), ethambutol (EMB) and streptomycin (STM) in human plasma using a hydrophilic interaction liquid chromatography-MS/MS (HILIC-MS/MS). In this study, phenformin and rifaximin were selected as the internal standards of PZA, INH, EMB, STM and RFP. Multiple reaction monitoring (MRM) was used for quantification. Sample extraction procedure is simple using protein precipitation with methanol containing 5mM ammonium acetate,0.1% formic acid and 10%water. After the optimization of the chromatographic conditions and MS/MS conditions, a HILIC column was selected for the separation and the mobile phase was composed of methanol and 0.1% formic acid 5mM ammonium acetate solution (65:35, v/v). All components were eluted within 2min under the optimum LC-MS/MS conditions. The recovery extraction was between 83.4% and 95.9%. Matrix effects were between 91.6% and 98.6%. The LLOQ of PZA, INH and RFP was 4.0 ng/mL. LLOQ of STM and EMB was 10.0 ng/mL and 0.5 ng/mL, respectively. The intra- and inter-day precision at three concentration levels of quality control samples were less than 8% and 9% with accuracy from -9.3% to 6.2%, and from -7.5% to 7.3%, respectively. The results indicate that the method was simple, rapid, sensitive, precise as well as accurate. The method was successfully applied to simultaneous determination of PZA, INH, RFP, STM and EMB in plasma obtained at 2h of 23 tuberculosis patients after oral administration of standard dose of PZA, INH, RFP and EMB, or injection of STM. The devised method offers a means of high-throughput and simultaneous therapeutic drug monitoring of anti-tuberculosis drugs.2. A rapid, specific and sensitive ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed and validated for the simultaneous determination of ropivacaine (Rop), bupivacaine (Bup), lidocaine (Lid) and dexamethasone (Dex) in human and rat plasma. Cocaine (Coc) and triamcinolone acetonide(Tri) were used as the internal standards of Rop, Bup, Lid and Dex, respectively. The analytes were extracted from plasma by simple protein precipitation and then separated on a Shim-pack XR-ODSⅡcolumn within 3.5 min using gradient elution. The mobile phase was composed of 0.1% formic acid water and acetonitrile. By adusting the elution program, Rop, Bup Lid and Coc(IS) were eluted before 2.6min while Dex and Tri(IS) were eluted after 2.6min. Then the mass detection was performed in positive electrospray ionization mode (ESI+) before 2.6min for the detection of Rop, Bup, Lid and Coc(IS), and in negative electrospray ionization mode (ESI-) after 2.6min for the detection of Dex and Tri(IS). This strategy can enhance the sensitivity and ensure enough data points for each peak. The lower limit of quantification (LLOQ) was 0.04 ng/mL for Rop and Bup, and 0.4 ng/mL for Lid and Dex. The intra- and inter-day precision at LLOQ and three concentration levels of quality control samples were less than 8.0% with accuracy between -7.9 and 8.7%. This method was validated and proved to good selectivity, sensitivity, accuracy, precision and reproducibility. The method was successfully applied to simultaneous determination of Rop, Bup, Lid and Dex in human and rat plasma.3. The essential oils extracted from Coriandrum sativum L. were analyzed by GC-MS coupled with chemometric resolution methods. Peak cluster A (8.843-9.032min) and B (12.725-13.060min) were selected as examples to illustrate the resolution procedure. Through the chemometric resolution methods, peak clusters were uniquely resolved into the pure chromatographic profiles and mass spectra of each component. Qualitative analysis was performed by comparing the pure mass spectra with those in the NIST 05 mass spectral library. Quantitative analysis was performed using the total volume integration method. A total of 118 constituents were detected, of which 104 were identified, accounting for 97.27% of the total content. In our study, (2E)-2-Dodecenal (18.28% of total content) was the most abundant compound, followed by 1-Dodecanol (8.59%), (Z)-14-Methyl-8-hexadecenal (7.25%), and Tetradecanal (6.34%). The other important compounds were 1-Decanol (5.39%) and Decanal (4.41%). The results were different from those reported in references. The results indicate that GC-MS combined with chemometric resolution methods can greatly enhance the capability of separation and the reliability of qualitative and quantitative results. 4. Solid phase microextraction (SPME) is a new extraction techniques of volatile compounds developed in recent years. It had several advantages:easy to operate, small amount of sample and solvent free. The volatile constituents from Huoxiang Zhengqi San was extracted using headspace solid-phase microextraction (HS-SPME), and then was detected by GC-MS. HS-SPME parameters (fiber type, equilibrium time extraction temperature and time, and desorption temperature and time) were investigated. The optimum extraction conditions were achieved on a PDMS/DVB/CAR (50/30μm) fiber under the optimum HS-SPME parameters (equilibrium time:20min, extraction temperature:80℃, extraction time:30 min, desorption time:5min and desorption temperature:220℃). The peak clusters were resolved into the pure chromatographic profiles and mass spectra of each component using the chemometric methods before qualitative and quantitative analysis. A total of 149 constituents were detected, of which 112 were identified, accounting for 77.9% of the total content. The most amount of the compound was Patchouli alcohol(15.3279%) followed by a-Bulnesene(8.7294%), Caryophyllene(5.6596%),α-Guaiene(6.25152%), D-Limonene(4.9283%) andβ-Humulene(3.6892%)。5. Steam distillation (SD) and simultaneous distillation-extraction (SDE) are still most used in quality control and constituent study of traditional Chinese medicine due to the simple device, easy operating and low consumption. However, they need long time for extraction and large amount of sample. Comparative study of HS-SPME、SD and SDE was performed to extract the volatile constituents of Huoxiang ZhengQi San. Though the intensity of the chromatography obtained from HS-SPME was much lower than those obtained from SD and SDE, the GC-MS chromatographic profiles of the three methods are in high similarity. The lower chromatographic intensity obtained from HS-SPME was because of the limited capacity of the SPME fiber and the small amount of sample. With the help of resolution of chemometric method, all the peaks obtained from HS-SPME, SD and SDE were resolved into the pure chromatographic profiles and mass spectra of each component for the further qualitative and quantitative analysis. Ninety-one components, accounting for 88.7% of the total content of HS-SPME, were in common with those obtained from SD and SDE. The common components were Patchouli alcohol with amount above 10% in the three methods, followed by Caryophyllene(above 5%),α-Bulnesene(above 4%), D-Limonene(above 3%), (β-Humulene(above 3%) andα-Guaiene(above 2%). The results indicate that the HS-SPME can extract the most constituents those the SD and SDE extracted. The HS-SPME is simple and rapid. It may be a good alternative method for SD and SDE for the quality control and constituent study of Huoxiang Zhengqi San and the other products of Huoxiang Zhengqi San.