Journal of Chromatography B, 971 (2014) 64–71
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of puerarin and its active metabolite in human plasma by UPLC-MS/MS: Application to a pharmacokinetic study Hyo-Rin Jung a , Sook-Jin Kim a , Seong-Ho Ham b , Jung-Hee Cho b , Yong-Bok Lee c , Hea-Young Cho a,∗ a
College of Pharmacy, CHA University, 59 Yatap-Ro, Bundang-gu, Seongnam-Si, Gyeonggi-Do 463-712, Republic of Korea Jeonnam Development Institute for Korean Traditional Medicine, Jeollanam-do 288, Republic of Korea c College of Pharmacy, Chonnam National University, 300 Yongbong-Dong, Buk-Gu, Gwangju 500-757, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 1 May 2014 Accepted 11 September 2014 Available online 22 September 2014 Keywords: Puerarin Daidzein Human UPLC-MS/MS Pharmacokinetics
a b s t r a c t A rapid, selective and sensitive ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry method about the simultaneous determination of puerarin and its major active metabolite, daidzein, in human plasma was developed and validated in order to investigate the pharmacokinetics (PKs) of Gegen after the usual oral dose administration to human. Chromatography was carried out on a Kinetex C18 column (2.1 mm × 50 mm, 1.7 m) using 0.05% acetic acid in water and 0.05% acetic acid in methanol as mobile phase with a gradient elution. Liquid–liquid extraction with ethyl acetate in acidic condition could remove the interference and minimize the matrix effect of human plasma. The lower limit of quantification in human plasma was 0.2 ng/mL for both of compounds, puerarin and daidzein. The calibration curves for puerarin and daidzein in human plasma were linear over all the concentration range of 0.2–100 ng/mL with correlation coefficients greater than 0.998. This assay procedure was successfully applied to the PKs of puerarin and daidzein, after the usual oral dose of Gegen extract powder (2.56 g, containing 9.984 mg puerarin) in human subjects. © 2014 Published by Elsevier B.V.
1. Introduction Gegen, the dried root of Pueraria lobata (Willd.) Ohwi, has been frequently described in East Asia for its antipyretic, antidiarrhetic, diaphoretic, antiemetic, and antialcoholic purposes alone [1] or in combination with other traditional medications, such as Sanqi (Panax notoginseng) [2] and Danshen (Salvia miltiorrhiza) [3]. In the last few years, Gegen powder and its extract has attracted globally growing attention as a supplement throughout many countries, such as United States, United Kingdom, and Australia [4]. The predominant constituents of Gegen extract are considered to be glucoside conjugates of isoflavonoids (O- and C-glucosides), such as puerarin and daidzin, which are mostly responsible for their pharmacological and biochemical responses [5,6]. Puerarin (7, 4 -dihydroxyisoflavone-8-glucopyranoside) as naturally occurring isoflavone-C-glucoside, isolated from the
∗ Corresponding author. Tel.: +82 31 780 5324; fax: +82 31 780 5305. E-mail address: [email protected] (H.-Y. Cho). http://dx.doi.org/10.1016/j.jchromb.2014.09.015 1570-0232/© 2014 Published by Elsevier B.V.
traditional Chinese herbal medicine Pueraria Radix (the root of the kudzu, Pueraria lobata), has been widely used in East Asia due to the protective effects on various diseases including cardiovascular, cerebrovascular, neurological and perlipidaemic disorders [6–9]. While puerarin is a very useful agent, previously reported information about its mechanism is scarce and studies about its acute and severe adverse effects after several weeks of therapy are limited [10]. Also, to understand the metabolism of puerarin and its pathway will importantly contribute to the understanding and elucidation of its therapeutic as well as toxic effects [6,8,11]. Prasain et al. [6] suggested that puerarin is hydrolyzed to daidzein by bacterial enzymes in the large intestine and subsequently reduced to dihydrodaidzein and equol. Because puerarin is absorbed from the gastrointestinal tract without being hydrolyzed, it is possible that puerarin is transferred to the portal circulation by the sodium-dependent glucose transporter 1. Because it may also be taken up by tissues such as in the liver, it can undergo oxidative metabolism involving certain cytochrome P450 isozymes. One of the routes of excretion of puerarin could be through the bile into the intestine and hence to the colon, where it is likely to be partly
H.-R. Jung et al. / J. Chromatogr. B 971 (2014) 64–71
metabolized by colonic microflora and then reabsorbed [6]. Therefore, the decision of dosage and administration is very important in the standardization of drug efficacy and toxicity. During the past decades, an increasing number of publications were reported about analytical methods of puerarin using highperformance liquid chromatography (HPLC) at different biological matrixes including plasma [12,13], cortex [14], urine, feces and organ tissues [15] in rats, dog plasma [16] and human plasma [17] after oral administration of puerarin. As well as HPLC, several studies applying liquid chromatography tandem mass spectrometry (LC-MS/MS) method have been reported for the quantification of puerarin tissue samples [9], plasma [10,18] and serum [19] in rats, or dog plasma [20]. Among them, Wang et al. [20] presented the LC-MS/MS method with a comparably higher sensitivity of relatively LLOQ 0.39 ng/mL than other studies [9,10,18,19] with a LLOQ of 2–18.6 ng/mL for puerarin. Also, further studies provided information about the simultaneous determination of puerarin and daidzein in rat plasma by using LC-MS [2], UPLC-MS [21] or UPLCMS/MS [22], serum [23] and urine [24] in humans by using HPLC. However, even though there are a lot of conventional analysis data, no previous publications have been reported describing the simultaneous quantification of puerarin and daidzein in human plasma by using LC-MS/MS after the usual oral dose administration of Puerariae Radix formulation to human until now. Also, although a lot of pharmacokinetic studies have been reported with regards to safflor yellow A or puerarin [25–30], no information has been reported about pharmacokinetic investigations on puerarin and daidzen, and simultaneous PKs of puerarin and daidzein in humans after the administration of Gegen preparations. Especially, an improved sensitivity is required in order to investigate the PKs of Gegen after the usual oral dose administration to human. Therefore, we aimed to develop a validated method with UPLC-MS/MS for the rapid, sensitive and accurate simultaneous determination of puerarin and its metabolite, daidzein in human plasma. Our established method was applied to investigate the PKs of puerarin and daidzein in humans after the usual oral dose administration of Gegen extract powder (2.56 g, containing 9.984 mg puerarin) to Korean subjects.
2. Materials and methods 2.1. UPLC-MS/MS analysis 2.1.1. Chemicals and reagents The reference standard of puerarin (purity >98.5%), daidzein (purity >97%) and topiramate (Fig. 1) as internal standard were purchased from Sigma–Aldrich (St. Louis, MO, USA). Pueraria root extract powder (Gegen extract powder) was produced in HanZung
65
Pharmaceutical Co., Ltd. (Daejeon, Korea), and kindly provided by Jeonnam Development Institute for Korean Traditional Medicine (Jeollanam-do, Korea). Methanol and ethyl acetate (HPLC grade) were supplied by J.T. Baker (Phillipsburg, NJ, USA). Acetic acid was purchased from Sigma–Aldrich (St. Louis, MO, USA). The other chemicals were of HPLC grade or highest quality available. HPLC grade water was obtained from a Milli-Q water purification system (Millipore Co., Milford, MA, USA) and used throughout this study. 2.1.2. Instrumentation and chromatographic conditions The liquid chromatography was performed on an AcquityTM UPLC system (Waters Corp., Milford, MA, USA) coupled with a Mass Spectrometer (XenoTM TQ-S, Waters Corp.). A 5 L aliquot of each sample was injected into a Phenomenex Kinetex C18 column (2.1 mm × 50 mm, 1.7 m particle size, Phenomenex, Torrance, CA, USA) with a column temperature of 23 ± 0.5 ◦ C. Gradient elution was employed for chromatographic separation using 0.05% acetic acid in water (solution A) and 0.05% acetic acid in methanol (solution B) at a flow rate of 0.2 mL/min. The gradient elution program was as follows: the initial mobile phase was maintained at 10% B for 0.5 min, linearly increased to 50% B over 1 min and held constant for up to 3 min, then linearly increased to 90% B for 1 min and finally returned to the initial condition (1 min). The mass spectrometer was operated with electrospray ionization (ESI) interface in a negative ion mode. Multiple reaction monitoring (MRM) transitions were used for quantification, which were m/z 415.1 → 267.0 for puerarin, m/z 253.2 → 223.0 for daidzein and m/z 338.1 → 77.9 for IS, respectively. The following parameters were employed: capillary voltage 2.6 kV, ion source temperature 150 ◦ C and desolvation temperature 350 ◦ C. Nitrogen was used as cone and desolvation gas at a flow rate of 150 and 600 L/h, respectively. The optimized collision energy was 35 eV for puerarin and daidzein. The cone voltage of puerarin and daidzein was 35 eV and 38 eV, respectively. Argon gas was employed at a pressure of approximately 4.2 × 10−3 mbar. Data acquisition and analysis were achieved using Masslynx 4.1 software (Waters Corp., Milford, USA). 2.1.3. Preparation of standards and quality control (QC) samples The standard stock solutions of puerarin, daidzein and internal standard (IS, topiramate) were prepared by dissolving accurately weighed standard compound in methanol at a concentration of 1 mg/mL and stored in a refrigerator (−20 ◦ C). The standard working solutions of mixed analytes were prepared by dilution with 50% methanol in water to achieve final concentrations of 2, 5, 10, 50, 100, 500 and 1000 ng/mL. The samples for the standard calibration curves were prepared by spiking 900 L of blank human plasma with 100 L of standard working solutions. The plasma concentration ranged from 0.2 to 100 ng/mL for puerarin and daidzein.
Fig. 1. Structures of the investigated compounds and internal standard topiramate. (A) puerarin, (B) daidzein, and (C) topiramate (IS).
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H.-R. Jung et al. / J. Chromatogr. B 971 (2014) 64–71
The IS working solution (20 ng/mL) was prepared in 50% methanol in water. The QC samples for puerarin and daidzein were individually prepared at four concentrations (0.2, 0.6, 16 and 80 ng/mL) for the evaluation of accuracy and precision. The calibration and QC samples were freshly prepared on the day of analysis. 2.1.4. Extraction procedures The liquid–liquid extraction (LLE) method was selected for the analysis. 100 L of human plasma was spiked with 10 L of IS (20 ng/mL in 50% methanol) solution and 100 L of acetic acid (0.1% in water) was added. The mixture solution was then extracted with 800 L of ethyl acetate by vigorous vortex-mixing for 3 min and centrifugation at 10,000 × g for 5 min at room temperature. 700 L of supernatant was transferred to clean tubes and evaporated to dryness under a gentle nitrogen stream at 50 ◦ C. The extraction residual was reconstituted with 300 L of 30% methanol in water and vortex-mixed for 1 min. After 5 min of centrifugation at 10,000 × g, 5 L aliquots were injected into the UPLC-MS/MS system. 2.1.5. Content of puerarin The determination of puerarin in Gegen extract powder was carried out according to the assay test for Pueraria Root Extract Powder in the Korean Pharmacopoeia (9th Edition) and Korean Herbal Pharmacopoeia IV. About 13 g of the powdered sample accurately weighed, added 50 mL of methanol, extracted under a reflux condenser for 3 h, and filtered. To the filtrates, added methanol to make exactly 100 mL. This solution was exactly taken 10 mL, added methanol to make 50 mL, filtered and used this solution as the test solution. Separately, about 10 mg of puerarin reference standard (RS) accurately weighed, dissolved in methanol to make exactly 100 mL, and used this solution as the standard solution. 10 L each of the test solution and the standard solution pipetted and performed the test as directed under HPLC. Puerarin content was analyzed by HPLC using -Bondapak C18 octadecylsilyl silica gel stainless column with a mixture of methanol and water (25:75) as mobile phase at UV 254 nm. The flow rate was 1.0 mL/min. The peak areas, AT and AS , of puerarin of the test solution and the standard solution were determined, respectively. Amount (mg) of puerarin (C21 H20 O9 ) = amount (mg) of puerarin RS × AT /AS × 5
2.2. Method validation 2.2.1. Specificity The specificity of the method was determined by evaluating the interferences of the endogenous compounds using screening analysis of standard puerarin and daidzein, drug-free plasma, plasma spiked with puerarin and daidzein, and plasma samples obtained after Gegen extract powder administration (2.56 g) to Korean volunteers. 2.2.2. Linearity and LLOQ Calibration curves for puerarin and daidzein were validated with a series of standard samples in the range of 0.2–100 ng/mL in human plasma. The linearity of the calibration curves constructed by weighted (1/concentration2 ) linear least-squares regression was determined by fitting the ratio of the peak area of the analytes to that of the IS versus the nominal concentration of the analytes. A straight-line regression equation was presented with its correlation coefficient (r). The sensitivity of the method was expressed as the lower limit of quantification (LLOQ) defined as the lowest concentration of
standard samples giving a signal-to-noise ratio of at least 10:1, in accordance with an acceptable accuracy between 80% and 120% of the theoretical value and a precision
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of puerarin and its active metabolite in human plasma by UPLC-MS/MS: Application to a pharmacokinetic study Hyo-Rin Jung a , Sook-Jin Kim a , Seong-Ho Ham b , Jung-Hee Cho b , Yong-Bok Lee c , Hea-Young Cho a,∗ a
College of Pharmacy, CHA University, 59 Yatap-Ro, Bundang-gu, Seongnam-Si, Gyeonggi-Do 463-712, Republic of Korea Jeonnam Development Institute for Korean Traditional Medicine, Jeollanam-do 288, Republic of Korea c College of Pharmacy, Chonnam National University, 300 Yongbong-Dong, Buk-Gu, Gwangju 500-757, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 1 May 2014 Accepted 11 September 2014 Available online 22 September 2014 Keywords: Puerarin Daidzein Human UPLC-MS/MS Pharmacokinetics
a b s t r a c t A rapid, selective and sensitive ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry method about the simultaneous determination of puerarin and its major active metabolite, daidzein, in human plasma was developed and validated in order to investigate the pharmacokinetics (PKs) of Gegen after the usual oral dose administration to human. Chromatography was carried out on a Kinetex C18 column (2.1 mm × 50 mm, 1.7 m) using 0.05% acetic acid in water and 0.05% acetic acid in methanol as mobile phase with a gradient elution. Liquid–liquid extraction with ethyl acetate in acidic condition could remove the interference and minimize the matrix effect of human plasma. The lower limit of quantification in human plasma was 0.2 ng/mL for both of compounds, puerarin and daidzein. The calibration curves for puerarin and daidzein in human plasma were linear over all the concentration range of 0.2–100 ng/mL with correlation coefficients greater than 0.998. This assay procedure was successfully applied to the PKs of puerarin and daidzein, after the usual oral dose of Gegen extract powder (2.56 g, containing 9.984 mg puerarin) in human subjects. © 2014 Published by Elsevier B.V.
1. Introduction Gegen, the dried root of Pueraria lobata (Willd.) Ohwi, has been frequently described in East Asia for its antipyretic, antidiarrhetic, diaphoretic, antiemetic, and antialcoholic purposes alone [1] or in combination with other traditional medications, such as Sanqi (Panax notoginseng) [2] and Danshen (Salvia miltiorrhiza) [3]. In the last few years, Gegen powder and its extract has attracted globally growing attention as a supplement throughout many countries, such as United States, United Kingdom, and Australia [4]. The predominant constituents of Gegen extract are considered to be glucoside conjugates of isoflavonoids (O- and C-glucosides), such as puerarin and daidzin, which are mostly responsible for their pharmacological and biochemical responses [5,6]. Puerarin (7, 4 -dihydroxyisoflavone-8-glucopyranoside) as naturally occurring isoflavone-C-glucoside, isolated from the
∗ Corresponding author. Tel.: +82 31 780 5324; fax: +82 31 780 5305. E-mail address: [email protected] (H.-Y. Cho). http://dx.doi.org/10.1016/j.jchromb.2014.09.015 1570-0232/© 2014 Published by Elsevier B.V.
traditional Chinese herbal medicine Pueraria Radix (the root of the kudzu, Pueraria lobata), has been widely used in East Asia due to the protective effects on various diseases including cardiovascular, cerebrovascular, neurological and perlipidaemic disorders [6–9]. While puerarin is a very useful agent, previously reported information about its mechanism is scarce and studies about its acute and severe adverse effects after several weeks of therapy are limited [10]. Also, to understand the metabolism of puerarin and its pathway will importantly contribute to the understanding and elucidation of its therapeutic as well as toxic effects [6,8,11]. Prasain et al. [6] suggested that puerarin is hydrolyzed to daidzein by bacterial enzymes in the large intestine and subsequently reduced to dihydrodaidzein and equol. Because puerarin is absorbed from the gastrointestinal tract without being hydrolyzed, it is possible that puerarin is transferred to the portal circulation by the sodium-dependent glucose transporter 1. Because it may also be taken up by tissues such as in the liver, it can undergo oxidative metabolism involving certain cytochrome P450 isozymes. One of the routes of excretion of puerarin could be through the bile into the intestine and hence to the colon, where it is likely to be partly
H.-R. Jung et al. / J. Chromatogr. B 971 (2014) 64–71
metabolized by colonic microflora and then reabsorbed [6]. Therefore, the decision of dosage and administration is very important in the standardization of drug efficacy and toxicity. During the past decades, an increasing number of publications were reported about analytical methods of puerarin using highperformance liquid chromatography (HPLC) at different biological matrixes including plasma [12,13], cortex [14], urine, feces and organ tissues [15] in rats, dog plasma [16] and human plasma [17] after oral administration of puerarin. As well as HPLC, several studies applying liquid chromatography tandem mass spectrometry (LC-MS/MS) method have been reported for the quantification of puerarin tissue samples [9], plasma [10,18] and serum [19] in rats, or dog plasma [20]. Among them, Wang et al. [20] presented the LC-MS/MS method with a comparably higher sensitivity of relatively LLOQ 0.39 ng/mL than other studies [9,10,18,19] with a LLOQ of 2–18.6 ng/mL for puerarin. Also, further studies provided information about the simultaneous determination of puerarin and daidzein in rat plasma by using LC-MS [2], UPLC-MS [21] or UPLCMS/MS [22], serum [23] and urine [24] in humans by using HPLC. However, even though there are a lot of conventional analysis data, no previous publications have been reported describing the simultaneous quantification of puerarin and daidzein in human plasma by using LC-MS/MS after the usual oral dose administration of Puerariae Radix formulation to human until now. Also, although a lot of pharmacokinetic studies have been reported with regards to safflor yellow A or puerarin [25–30], no information has been reported about pharmacokinetic investigations on puerarin and daidzen, and simultaneous PKs of puerarin and daidzein in humans after the administration of Gegen preparations. Especially, an improved sensitivity is required in order to investigate the PKs of Gegen after the usual oral dose administration to human. Therefore, we aimed to develop a validated method with UPLC-MS/MS for the rapid, sensitive and accurate simultaneous determination of puerarin and its metabolite, daidzein in human plasma. Our established method was applied to investigate the PKs of puerarin and daidzein in humans after the usual oral dose administration of Gegen extract powder (2.56 g, containing 9.984 mg puerarin) to Korean subjects.
2. Materials and methods 2.1. UPLC-MS/MS analysis 2.1.1. Chemicals and reagents The reference standard of puerarin (purity >98.5%), daidzein (purity >97%) and topiramate (Fig. 1) as internal standard were purchased from Sigma–Aldrich (St. Louis, MO, USA). Pueraria root extract powder (Gegen extract powder) was produced in HanZung
65
Pharmaceutical Co., Ltd. (Daejeon, Korea), and kindly provided by Jeonnam Development Institute for Korean Traditional Medicine (Jeollanam-do, Korea). Methanol and ethyl acetate (HPLC grade) were supplied by J.T. Baker (Phillipsburg, NJ, USA). Acetic acid was purchased from Sigma–Aldrich (St. Louis, MO, USA). The other chemicals were of HPLC grade or highest quality available. HPLC grade water was obtained from a Milli-Q water purification system (Millipore Co., Milford, MA, USA) and used throughout this study. 2.1.2. Instrumentation and chromatographic conditions The liquid chromatography was performed on an AcquityTM UPLC system (Waters Corp., Milford, MA, USA) coupled with a Mass Spectrometer (XenoTM TQ-S, Waters Corp.). A 5 L aliquot of each sample was injected into a Phenomenex Kinetex C18 column (2.1 mm × 50 mm, 1.7 m particle size, Phenomenex, Torrance, CA, USA) with a column temperature of 23 ± 0.5 ◦ C. Gradient elution was employed for chromatographic separation using 0.05% acetic acid in water (solution A) and 0.05% acetic acid in methanol (solution B) at a flow rate of 0.2 mL/min. The gradient elution program was as follows: the initial mobile phase was maintained at 10% B for 0.5 min, linearly increased to 50% B over 1 min and held constant for up to 3 min, then linearly increased to 90% B for 1 min and finally returned to the initial condition (1 min). The mass spectrometer was operated with electrospray ionization (ESI) interface in a negative ion mode. Multiple reaction monitoring (MRM) transitions were used for quantification, which were m/z 415.1 → 267.0 for puerarin, m/z 253.2 → 223.0 for daidzein and m/z 338.1 → 77.9 for IS, respectively. The following parameters were employed: capillary voltage 2.6 kV, ion source temperature 150 ◦ C and desolvation temperature 350 ◦ C. Nitrogen was used as cone and desolvation gas at a flow rate of 150 and 600 L/h, respectively. The optimized collision energy was 35 eV for puerarin and daidzein. The cone voltage of puerarin and daidzein was 35 eV and 38 eV, respectively. Argon gas was employed at a pressure of approximately 4.2 × 10−3 mbar. Data acquisition and analysis were achieved using Masslynx 4.1 software (Waters Corp., Milford, USA). 2.1.3. Preparation of standards and quality control (QC) samples The standard stock solutions of puerarin, daidzein and internal standard (IS, topiramate) were prepared by dissolving accurately weighed standard compound in methanol at a concentration of 1 mg/mL and stored in a refrigerator (−20 ◦ C). The standard working solutions of mixed analytes were prepared by dilution with 50% methanol in water to achieve final concentrations of 2, 5, 10, 50, 100, 500 and 1000 ng/mL. The samples for the standard calibration curves were prepared by spiking 900 L of blank human plasma with 100 L of standard working solutions. The plasma concentration ranged from 0.2 to 100 ng/mL for puerarin and daidzein.
Fig. 1. Structures of the investigated compounds and internal standard topiramate. (A) puerarin, (B) daidzein, and (C) topiramate (IS).
66
H.-R. Jung et al. / J. Chromatogr. B 971 (2014) 64–71
The IS working solution (20 ng/mL) was prepared in 50% methanol in water. The QC samples for puerarin and daidzein were individually prepared at four concentrations (0.2, 0.6, 16 and 80 ng/mL) for the evaluation of accuracy and precision. The calibration and QC samples were freshly prepared on the day of analysis. 2.1.4. Extraction procedures The liquid–liquid extraction (LLE) method was selected for the analysis. 100 L of human plasma was spiked with 10 L of IS (20 ng/mL in 50% methanol) solution and 100 L of acetic acid (0.1% in water) was added. The mixture solution was then extracted with 800 L of ethyl acetate by vigorous vortex-mixing for 3 min and centrifugation at 10,000 × g for 5 min at room temperature. 700 L of supernatant was transferred to clean tubes and evaporated to dryness under a gentle nitrogen stream at 50 ◦ C. The extraction residual was reconstituted with 300 L of 30% methanol in water and vortex-mixed for 1 min. After 5 min of centrifugation at 10,000 × g, 5 L aliquots were injected into the UPLC-MS/MS system. 2.1.5. Content of puerarin The determination of puerarin in Gegen extract powder was carried out according to the assay test for Pueraria Root Extract Powder in the Korean Pharmacopoeia (9th Edition) and Korean Herbal Pharmacopoeia IV. About 13 g of the powdered sample accurately weighed, added 50 mL of methanol, extracted under a reflux condenser for 3 h, and filtered. To the filtrates, added methanol to make exactly 100 mL. This solution was exactly taken 10 mL, added methanol to make 50 mL, filtered and used this solution as the test solution. Separately, about 10 mg of puerarin reference standard (RS) accurately weighed, dissolved in methanol to make exactly 100 mL, and used this solution as the standard solution. 10 L each of the test solution and the standard solution pipetted and performed the test as directed under HPLC. Puerarin content was analyzed by HPLC using -Bondapak C18 octadecylsilyl silica gel stainless column with a mixture of methanol and water (25:75) as mobile phase at UV 254 nm. The flow rate was 1.0 mL/min. The peak areas, AT and AS , of puerarin of the test solution and the standard solution were determined, respectively. Amount (mg) of puerarin (C21 H20 O9 ) = amount (mg) of puerarin RS × AT /AS × 5
2.2. Method validation 2.2.1. Specificity The specificity of the method was determined by evaluating the interferences of the endogenous compounds using screening analysis of standard puerarin and daidzein, drug-free plasma, plasma spiked with puerarin and daidzein, and plasma samples obtained after Gegen extract powder administration (2.56 g) to Korean volunteers. 2.2.2. Linearity and LLOQ Calibration curves for puerarin and daidzein were validated with a series of standard samples in the range of 0.2–100 ng/mL in human plasma. The linearity of the calibration curves constructed by weighted (1/concentration2 ) linear least-squares regression was determined by fitting the ratio of the peak area of the analytes to that of the IS versus the nominal concentration of the analytes. A straight-line regression equation was presented with its correlation coefficient (r). The sensitivity of the method was expressed as the lower limit of quantification (LLOQ) defined as the lowest concentration of
standard samples giving a signal-to-noise ratio of at least 10:1, in accordance with an acceptable accuracy between 80% and 120% of the theoretical value and a precision
