SINGLE LABORATORY VALIDATION OF UHPLC-MS/MS ASSAY OF RED CLOVER Ruth N. Muchiri,1 and Richard B. van Breemen1 1Linus Pauling Institute, Oregon State University, 2900 SW Campus Way, Corvallis, Oregon USA. OBJECTIVES RESULTS • Develop and validate UHPLC-MS/MS assay for red clover • Base line separation of all 6 red clover isoflavones was obtained in 5 min during UHPLC • All analytes were stable in the autosampler (10 °C) for 24 h (covers the time for injection and isoflavones in dietary supplements and human serum • No interfering peaks were observed at the same retention times as the isoflavones (Figure 3) UHPLC-MS/MS of all analytes processed at any specific validation study (Table 3). • Apply the validated method to standardize red clover extract and • The standard curves were linear from the LLOQ to the ULOQ concentrations for all compounds • Freeze-thaw cycle of analytes in human serum matrix post extraction were stable after 2 cycles. 2 dietary supplement products, and analyze serum samples from with a R >0.999. The standards were spiked in human serum matrix (Figure 4 & Table 1). • The QCs were within the acceptance criteria: Below 15% for L, M, and H; and below 20% at LLOQ. clinical trials • The mean recovery (based on low, medium and high QC (quality control) standards ranged • The intraday and interday coefficients of variation obtained from all the isoflavones were <13% between 80 and 108% in serum matrix (Table 2). except for daidzein at low concentration (Table 4). Table 3. Stability studies of red clover isoflavones (%). BACKGROUND A Figure 4. An example of QC (Medium) Autosampler With their origins in traditional medicine, botanical dietary supplements standard curve that was Freeze thaw (×1) Freeze thaw (×2) remain in widespread use. For example, preparations of red clover used in the quantification n ≥ 3 (24 h) (Trifolium pratense L.) are currently used to treat respiratory problems of genistein in QC samples. The standard Genistein 104 ± 3.6 97.9 ± 4.3 84.2 ± 17.2 (asthma, whooping cough and bronchitis), skin disorders (eczema and curves were fit by linear psoriasis), and menopausal issues in women, especially vasomotor regression without the use Daidzein 105 ± 8.2 97.5 ± 3.6 94.4 ± 6.4 symptoms [1-2]. The active isoflavones are concentrated in the aerial of weighing factor. parts of red clover. Daidzein and genistein are estrogenic, whereas Biochanin A 104 ± 5.6 98.0 ± 5.4 79.3 ± 28.1 biochanin A and formononetin can be metabolized to genistein and Table 1. Accuracy (%) of genistein standards daidzein, respectively, and are considered proestrogens. In contrast, B Irilone 93.9 ± 5.7 91.9 ± 13.5 70.1 ± 3.1 Genistein ng/mL Accuracy[%] the isoflavones irilone and prunetin in red clover have progesterone 0.6 108.5 ± 7.7 activity [3]. See chemical structures in Figure 1. Prunetin 105 ± 4.2 90.6 ± 9.4 77.8 ± 28.4 1.8 107.8 ± 8.3 For safe use and to ensure reproducible effects, red clover dietary 5.4 96.9 ± 3.9 Formononetin 103 ± 4.1 101 ± 1.4 78.5 ± 19.3 supplements should be chemically standardized to the active 16.2 107.4 ± 1.6 Table 4. Interday and intraday accuracy and precision. isoflavones. In this study, a fast UHPLC-MS/MS based method was 48.6 95.5 ± 3.3 Concentration developed and validated for the quantitative of all 6 isoflavones in red 145.8 100.8 ± 1.9 Range Genistein Daidzein Biochanin A Irilone Prunetin Formononetin clover dietary supplements using deuterium-labelled internal standards 437.4 100.0 ± 2.4 (n = 10) (Figure 1) to correct for variations during sample preparation and C Table 2. Recovery of extracted standards in human serum matrix LLOQ 0.3 0.2 0.1 0.3 0.1 0.1 analysis. Additionally, the UHPLC-MS/MS assay was validated using aCV% 0.9 6.4 0.8 1.1 0.4 0.9 human serum matrix with the goal of supporting clinical research where Concentration bCV% 0.8 6.3 0.5 1.5 0.4 1.0 Range Genistein Daidzein Biochanin A Irilone Prunetin Formononetin serum samples will be measured for the various red clover isoflavones. (n = 5) Low (ng/mL) 0.9 0.6 0.3 0.9 0.3 0.3 aCV% 1.0 26 3.0 1.9 1.2 3.5 References bCV% 0.8 28 3.8 0.5 0.8 2.8 Low (ng/mL) 0.9 0.6 0.3 0.9 0.1 0.3 1. Kolodziejczyk-Czepas, J. J. Ethnopharmacol. 2012, 143, 14. Recover % 108.4 99.6 105.6 106.5 95.6 98.2 Medium 16.2 5.4 5.4 16.2 5.4 1.8 (ng/mL) 2. Piersen, C. E.; Booth, N. L.; Sun, Y.; Liang, W.; Burdette, J. E.; van Breemen, 3.1 4.5 5.9 12.8 4.9 8.5 Medium aCV% R. B. v.; Geller, S. E.; Gu, C.; Banuvar, S.; Shulman, L. P.; Bolton, J. L.; 16.2 5.4 5.4 16.2 5.4 1.8 3.4 4.8 5.2 4.3 4.3 9.8 (ng/mL) bCV% Farnsworth, N. R. Curr. Med. Chem. 2004, 11, 1361. 100.6 96.6 98.4 82.2 98.9 99.4 Recovery % Figure 3. UHPLC-MS/MS chromatograms of red clover isoflavones. High (ng/mL) 437.4 145.8 145.8 437.4 145.8 48.6 3. Lee, J.-H.; Dean, M.; Austin, J. R.; Burdette, J. E.; Murphy, B. T. J. Nat. Prod. A) Commercially available standards spiked into human serum, extracted through aCV% 4.9 4.6 6.8 11.5 7.0 5.4 2018, 81, 1962.) protein precipitation, dried under vacuum and reconstituted in equimolar High (ng/mL) 437.4 145.8 145.8 437.4 145.8 48.6 bCV% 5.9 2.6 4.2 3.9 4.1 2.3 methanol/water (0.01% formic acid). B) Isoflavones from a methanol extract of red Recovery % 100.1 94.5 94.5 78.4 94.0 95.4 clover dietary supplement capsule (1ug/mL). C) Blank serum aInterday, bIntraday. METHODS METHODS AND INSTRUMENTATION CONCLUSIONS • Red clover isoflavones were purchased at >98% purity (Figure 1) • Internal standard (50 ng/mL) • Ice cold methanol/ethanol • Dry supernatant under vacuum • Analyze 3 µL aliquot • A UHPLC-MS/MS assay for the analysis of red clover isoflavones was • Human serum (100 µL) (1:1, v/v), 800 µL • Reconstitute in 200 µL of using UPLC-MS/MS developed and validated • Recovery, quality control and stability assays were carried out through water/CH CN (0.01% formic (Shimadzu 8060 QQQ) • QC or standard curve working • Vortex on high (2 min) 3 • The standard curves were linear for both low and high isoflavone protein precipitation as outlined in Figure 2 solution • Spin (18,000×g, 30 min, 4 °C) acid) concentrations, recovery was excellent, and the isoflavones were Figure 2. Red clover isoflavones were extracted using equimolar ice cold methanol and ethanol, dried and reconstituted in 1:1 water and stable for the duration of the assay and as well as storage. acetonitrile containing 0.01% formic acid • The UHPLC-MS/MS assay is applicable to the standardization of red clover supplements and analysis of serum samples from clinical trials. • Red clover dietary supplement was extracted with 25 mg/mL methanol/ethanol, vortexed on high for 10 min, filtered, and the filtrate was dried under a stream of nitrogen FUTURE DIRECTION • 1 µg/mL solution of extract was prepared by adding internal standard (50 ng/mL) and equimolar water/acetonitrile (containing 0.01% formic acid) to 200 µL total • Apply the UHPLC-MS/MS assay for the analysis of commercial red • Samples were analyzed on a Shimadzu LCMS-8060 triple quadrupole mass spectrometer equipped with a Nexera UHPLC system (Kyoto, clover supplements. Japan) and an ACE Super C18 column (1.7 μm, 90 Å, 2.1 mm x100 mm). • UHPLC method: 20 to 55% acetonitrile (mobile phase B) in water (0.01% formic acid, mobile phase A) over 2 min; hold at 55% B for 2 min; ACKNOWLEDGEMENTS 90% B for 0.5 min; and re-equilibrate at 20% B for 1 min. Flow rate was 0.4 mL/min. This project was funded by grant P50AT000155 administrative supplement from the NIH National Center for Complementary and Integrative Health. • Shimadzu LabSolutions software version 5.6 was used for data analysis Figure 1. Chemical structures of red clover isoflavones and deuterated internal standards Authors thank Shimadzu for providing the UHPLC-MS/MS system used in this study..