| Part A. Simultaneous determination for six Aconitum alkaloids in Aikeqing 2 Capsules by LC-MS/MS techniquesObjective:Liquid Chromatography/Spectrometry method has been applied to study two aspests in this papper:1. To study types of aconitumAlkaloids within Aikeqing 2 Capsules by Liquid Chromatography/Ion Trap Mass Spectrometry (LC-MS") method.2. To establish quantification method ofaconitine, hypaconitine, mesaconitine, benzoylaconitine, benzoylhypaconitine, benzoylmesaconitine within Aikeqing 2 Capsules by High Performance Liquid Chromatography Tandem Mass method.Methods:1.To qualitative analyze aconitum Alkaloids in Aikeqing 2 Capsules, ca psule content was dissolved by 1% formic acid-acetonitrile solution, alka lized by 5% NaOH solution, extracted by diethyl ether. Chromatographic Sepa ration was perform by Waters Symmetry C18(4.6 mm × 150 mm,5 μm) column and a gradient elution of 10 mmol/L ammonium formate-acetonitrile and 0. 2% formic acid in water and acetonitrile was used as the mobile phase. The flow rate was set at 0.6 mL·min-1. Under the positive-ion electrospray i onization, full scan mode and automatic secondary stage MS scan mode has b een applied to obtain the mass data.2.The Quantitative detection of six aconitum Alkaloids in Aikeqing 2 Capsules was based on Liquid Chromatography Tandem Mass method(LC-MS/MS). Capsule content was ultrasonic extracted by 1% formic acid-acetonitrile s olution. The separation was performed on an Agilent Poroshell 120 EC-C18(2. 1 mm ×100 mm,2.7 μm) column and a gradient elution of 10 mmol/L ammoni um formate-acetonitrile and 0.2% formic acid in water and acetonitrile wa s used as the mobile phase. The flow rate was set at 0.3 mL·min-1.The dete ction of 6 targeted compounds was performed by positive-ion electrospray ionization mass spectrometry(ESI MS) under multiple reaction monitoring (MRM) mode. The transit ions of diester diterpenoid alkaloids and monoeste r-diterpenoid alkaloids executed as following:m/z 646.3→368.1/568.1 for Aconitine, m/z m/z 616.0→524.0/556.2 for Hypaconitine, m/z 632.2→104.8/ 572.0 for Mesaconitine, m/z 604.2→554.2/104.8 for Benzoylaconitine, m/z 5 74.1→104.9/542.1 for Benzoylhypaconitine,m/z 590.1→540.1/104.9 for Benz oylmesaconitine.Results:1. The quasi-molecular ion peak and characteristic fragment ions of ea ch component was obtained from Liquid Chromatography/Ion Trap Mass Spectr ometry(LC-MSn),under the full scan mode and auto MS2 mode. By analyze the mass data and spectrum, different types of aconitum Alkaloids in Aikeqing 2 Capsules has been identified. According to the mass data, Fuziline, Neolin e, Talatizamine,Chasmanine, Aconitine, Hypaconitine,Mesaconitine, Benzoylaco nitine, Benzoylhypaconitine, Benzoylmesaconitine was identified from Aikeq ing 2 Capsules.2. Under the the multi-reaction monitoring mode (MRM), the Aconitine, H ypaconitine, Mesaconitine, Benzoylaconitine, Benzoylhypaconitine, Benzoyl mesaconitine has been simultaneous determinated by High Performance Liqui d Chromatography Tandem Mass method in the Aikeqing 2 Capsules. The linear ranges were 0.5-500.0 ng · mL-1 (r=0.9999) for aconitine,0.5-500.0 ng · mL-1 (r=0.9996) for hypaconitine,0.5-500.0 ng · mL-1 (r=0.9993) for mesaconitin e.100.0-500.0 ng · mL-1 (r=0.9994) for benzoylaconitine,500.0-1000.0 ng · mL-1 (r=0.9995) for benzoylhypaconitine,500.0-1000.0 ng · mL-1 (r=0.9994) for benzoylmesaconitine. The average recoveries(n=9) were 104.29% for aconiti ne,99.87% for hypaconitine,96.81% for mesaconitine,98.74% for benzoylacon itine,98.77% for benzoylhypaconitine,96.20% for benzoylmesaconitine, The 1 imits of detection(LOD, S/N≥3) and quantitation(LOQ, S/N≥10) were 0. 024-0.044 ng/mL and 0.082-0.149 ng/mL.Conclusions:1.10 different types of aconitumAlkaloids in Aikeqing 2 Capsules has been identified by Liquid Chromatography/Ion Trap Mass Spectrometry(LC-MS n) method.2. A LC-MS/MS method has been established and applied for simultaneous quantification ofaconitine, hypaconitine, mesaconitine, benzoylaconitine, benzoylhypaconitine, benzoylmesaconitine within Aikeqing 2 Capsules.Part B. Simultaneous determination for four antidiabetic chemicals illegally added in a Chinese herbal health-care product Yangyi Pingtang Capsules by LC-MS/MS techniquesObjective:To establish an LC-MS/MS method for simultaneous quantification of metformin, phenformin, rosiglitazone, and glibenclamide in the Yangyipingtang Capsules.Methods:Under the the multi-reaction monitoring mode (MRM),the metformin, phenformin, rosiglitazone, and glibenclamide has been simultaneous determinated by High Performance Liquid Chromatography Tandem Mass method in the Yangyipingtang Capsules.Results:A HPLC-MS/MS method for simultaneous quantification of metformin, phenformin, rosiglitazone, and glibenclamide, four antidiabetics illegally added into a health-care food was developed. The separation was performed on a Thermo BDS Hypersil C8 column (2.1 mm×100 mm,2.4 μm) by gradient elution with the mixture of 0.2% aqueous formic acid (A) and acetonitrile-methanol (6:4, v/v) (B) as mobile phase. The flow rate was set at 0.3 mL · min-1. The detection of 4 target chemicals was performed by positive-ion electrospray ionization mass spectrometry (ESI-MS) in multiple-reaction monitoring (MRM) mode. The transitions of four antidiabetics executed as following:m/z 129.9 → 70.9 for metformin, m/z 205.9→9.9 for phenformin, m/z 357.9→134.9 for rosiglitazone, m/z 494.0→368.8 for glibenclamide.The limits of detection (LOD,S/N≥3)and quantitation(LOQ,S/N≥10)were 0.06～0.5 ng·mL-1 and 0.21～1.62 ng· mL-1, respectively. The average recoveries of the four analytes at three spiked concentration levels were 66.3% to 110.2% with relative standard deviations (RSDs) at 2.3%～12.2%Conclusions:A LC-MS/MS method has been established and applied for simultaneous quantification of metformin, phenformin, rosiglitazone, and glibenclamide in a Chinese herbal health-care product "Yangyipingtang" Capsules.
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