Rapid and Sensitive Quantification of Ursolic Acid and Oleanolic Acid in Human Plasma Using Ultra-performance Liquid Chromatography–Mass Spectrometry

Rapid and Sensitive Quantification of Ursolic Acid and Oleanolic Acid in Human Plasma Using... Abstract Ultra-performance liquid chromatography (UPLC) interfaced with atmospheric pressure chemical ionization mass–spectrometry was used to separate and quantify ursolic acid (UA) and oleanolic acid (OA) in human plasma. UA and OA were extracted from 0.5 mL human plasma using supported liquid extraction and separated utilizing an Acquity UPLC HSS column. The method has been validated for both UA and OA quantitation with a limit of detection of 0.5 ng/mL. The UPLC separations are carried out with isocratic elution with methanol and 5 mM ammonium acetate in water (85:15) as a mobile phase at a flow rate of 0.4 mL/min. The assay was linear from 1 ng/mL to 100 ng/mL for both analytes. The total analysis time was 7 min with the retention times of 3.25 (internal standard), 3.65 (UA) and 3.85 min (OA). Recovery of drug from plasma ranged from 70% to 115%. Analysis of quality control samples at 3, 30 and 80 ng/mL (n = 14) had an intra-day coefficient of variation of 9.9%, 4.3% and 5.5%, respectively. A proof-of-concept study in human patients who consumed apple peels indicates that this analytical method could be applied to clinical studies of UA and/or OA in human subjects. Introduction Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid; UA) and its isomer, oleanolic acid (3β-hydroxy-olea-12-en-oic acid; OA) (Figure 1) exist in natural plants and have been shown to exhibit anti-inflammatory, antifungal, antitumor and hepatoprotective pharmacological properties (1). Health-promoting effects of plants containing UA had been known for many centuries (2, 3) and recent reports of antitumor activity of the UA and OA (4) generated a wide interest in these compounds. In addition to the pharmacological properties listed above, it has recently been shown that UA reduces muscle atrophy, promotes muscle hypertrophy, and increases energy expenditure, leading to reduced obesity, improved glucose tolerance and decreased hepatic steatosis in mice (5, 6). These promising preclinical data suggest further studies in humans are warranted as UA and/or structural analogs might be useful therapeutic agents for a number of increasingly common metabolic disorders, including skeletal muscle atrophy, obesity, type 2 diabetes and non-alcoholic fatty liver disease (5, 6). Figure 1. View largeDownload slide Structures of ursolic acid, oleanolic acid and betulinic acid. Figure 1. View largeDownload slide Structures of ursolic acid, oleanolic acid and betulinic acid. UA and OA are very similar in structure, co-exist in some natural products such as apple peels, and share some common pharmacological properties. There are reports in the literature addressing isolation, separation and purification of these isomers as well as studies on their beneficial effects. The separation of these compounds still remains challenging (7). Different analytical methods have been used to separate and detect the two isomers (8–11). High performance liquid chromatography (HPLC) with ultraviolet (UV) detection (12–15), mass spectrometry (MS) (9, 16–19) or evaporative light scattering detectors (20) are the most common methods used for UA quantification in the biological samples. However, the need for a rapid and reproducible method with improved sensitivity is needed to conduct pharmacokinetic studies of UA where very low concentrations are expected. MS provides superior sensitivity compared with an UV and other detection methods, which is essential for detecting trace concentrations of UA in the plasma samples. Also, MS detection does not require extensive sample preparation, such as derivatization, and provides high resolution and rapid analysis. In this work, a method for the analysis of UA separated from OA in from plasma was developed and validated using ultra-performance liquid chromatography equipped with atmospheric pressure chemical ionization mass spectrometry (UPLC–MS). To our knowledge, this is the most sensitive and rapid method for UA detection in human plasma samples. More importantly, it also allows for the rapid separation of UA from OA, which is particularly important since both compounds are commonly found in natural products. This method has been used to support clinical studies of ursolic and OA in human subjects. Experimental Materials and reagents UA, OA, betulinic acid (Figure 1) and HPLC grade dichloromethane were purchased from Sigma-Aldrich Corp. (St Louis, MO, USA). Optima HPLC grade methanol, hexanes and American Chemical Society grade glacial acetic acid were purchased from Fisher Scientific (Pittsburgh, PA). Ammonium acetate (analytical reagent grade) was purchased from Mallinckrodt Baker Inc. (Paris, KY). HPLC grade water was from a MilliQ Integral water purification system (Millipore Corp., Bedford, MA). Human plasma was obtained from the DeGowin Blood Center at the University of Iowa Hospitals and Clinics. Stock and sample preparation To prepare plasma standards, UA and OA were dissolved separately in methanol to make 0.2 mg/mL stock solutions. About 5 μL of each solution was combined with 990 μL of methanol to form a concentrated working standard of 1 μg/mL containing both UA and OA. This standard was further diluted with methanol to 100 ng/mL to form a less concentrated working standard. Calibration standards were prepared by adding between 5 μL and 50 μL of the appropriate working standard to sufficient human plasma to prepare 0.5 mL of standards in a 2-mL Eppendorf tubes. The standard curve consisted of samples containing 1, 2, 4, 10, 20, 50 and 100 ng/mL of UA and OA. Control samples were 3, 30 and 80 ng/mL. Internal standard (IS) solution was prepared by dissolving betulinic acid to 0.2 mg/mL in methanol, and then further diluting it with methanol to 1 μg/mL. A solution containing acetic acid (4%) in water (450 μL) was added to each sample along with 10 μL of the internal standard. Blanks and controls were prepared in the same manner. All solutions were mixed and subjected to supported liquid extraction (SLE) prior to instrumental analysis. Sample extraction Isolute SLE+ 6 mL (1 mL sample volume) SLE cartridges (Part# 820-0140-C, Biotage AB, Uppsala, Sweden) were used for sample preparation. A Cerex SPE processor from Varian (Palo Alto, CA) using nitrogen gas to modulate flow was used. Samples were transferred into SLE+ cartridges and a nitrogen flow was applied for 15 s to help load the samples. The samples were allowed to adsorb onto the packing material for 10 min. The analytes were eluted with a 5-mL solvent mixture of hexanes and dichloromethane (2:1 volume:volume) under gravity flow for ~15 min. Nitrogen flow was applied to maximize sample elution for 10 s at the end. The eluates were collected in 13 mm×100 mm glass tubes and solvent was evaporated under flowing nitrogen at 25°C. The residue was reconstituted in 200 μL of 5 mM ammonium acetate in water (15%) and methanol (85%) and transferred to a 2-mL microcentrifuge tube. The samples were centrifuged at 14,000 rpm at 7°C for 30 min (Mikrocentrifuge 200 R, Hettich AG, Bäch, Swizerland). The centrifugates were filtered through a 13-mm 0.2-μm syringe filter (Life Sciences Acrodisc LC) using 1-mL plastic syringe into HPLC injection vials. Instrumentation and chromatographic conditions The instrumentation system consisted of a Waters Acquity UPLC system equipped with binary solvent manager, sample manager and column manager operating under Empower software (Waters Corporation, Milford, MA, USA) and a Shimadzu 2010A LC–MS platform in atmospheric pressure chemical ionization (APCI) negative ion mode using selected ion monitoring and operating under LC–MS Solution software (Version 2.04H3, Shimadzu, Columbia, MD, USA). An Acquity UPLC HSS analytical column, C-18, 1.8 μm, 2.1 mm × 100 mm (Waters Corporation, Milford, MA, USA) preceded by a Phenomenex C-18 ultra UHPLC security guard column was used. Separation conditions were: 7°C sample temperature; room column temperature; sample was injected in partial loop with overfill injection mode; 15 μL injection volume. Isocratic analysis was conducted at a 0.4-mL/min flow rate. Mobile phase A (15%) was 5 mM ammonium acetate; mobile phase B (85%) was methanol. The total run time for a LC–MS analysis was 7 min. The mass spectrometer was tuned using a polyethylene glycol solution following the manufacturer’s protocol. The scan interval was 0.3 s, microscan 0.1 amu, APCI temperature was 450°C, the curved desolvation line temperature (200°C), and the heat block temperature (200°C). Nitrogen flow rate was 2.5 L/min; detector voltage was 1.6 kV. The monitored mass-to-charge ratio for all three analytes was 455.45. The Waters Acquity system was configured to send a signal via a contact closure to the MS system to start data acquisition following sample injection. This allowed the Waters UPLC system to be used for sample injection and bioanalytical separation, and the Shimadzu MS to acquire the mass spectrum data even when the instruments do not normally work together in a single system. Method validation Specificity and selectivity The specificity of the method was evaluated by analyzing six different sources of blank plasma samples. Blank plasma samples were compared with spiked plasma samples at the linearity and lower limits of quantitation (LLOQ) level and the response of any co-eluting interferences calculated. Linearity and lower limits of quantitation The calibration curves were prepared by spiking blank plasma samples at seven concentrations, from 1 ng/mL to 100 ng/mL. The linearity of each calibration curve was determined by plotting the peak-area ratio of the analytes to IS versus the nominal concentration with weighted (1/concentration) least square regression. The LLOQ for the analytes were the lowest concentration with signal-to-noise ≥ 5, which could be quantitatively determined with precision and accuracy ≤ 20%, evaluated by analyzing samples in six replicates. Precision and accuracy Precision and accuracy were assessed by analyzing quality control (QC) samples in six replicates at low, medium and high concentrations of the analytes in six replicates on the same day and on three different days, respectively. Precision was defined as relative standard deviation of the measured concentration and the accuracy as the relative error of the measured mean value from the nominal value. Stability studies have previously been reported for UA and OA and are not repeated in this study (15). Results The mass spectrometric conditions were initially optimized by injecting standards at concentration of 1 μg/mL and adjusting MS voltages to improve analyte signal. The parent ion spectra for UA, OA and IS were monitored in the both positive and negative ionization with APCI and electrospray ionization (ESI) (data not shown). Due to the carboxyl in the chemical structures of UA, OA and IS, the full-scan mass spectrum showed that the signal intensity in the negative ion mode was much higher than that in the positive APCI ion mode. UA, OA and IS of endogenous compounds, are isobaric with very similar physio-chemical properties and share the same mass. Therefore, MS specificity by itself is not always adequate to separate all analytes, so we have chromatographically separated them (Figure 2). Figure 2. View largeDownload slide UPLC chromatograms of blank plasma sample (A), low standard sample (1 ng/ml) (B), middle standard sample (10 ng/ml) (C), and an unknown sample (D). Detection m/z is 455.45. Ursolic acid retention time is 3.85 min, oleanolic acid – 3.65 min., betulinic acid – 3.25 min. Figure 2. View largeDownload slide UPLC chromatograms of blank plasma sample (A), low standard sample (1 ng/ml) (B), middle standard sample (10 ng/ml) (C), and an unknown sample (D). Detection m/z is 455.45. Ursolic acid retention time is 3.85 min, oleanolic acid – 3.65 min., betulinic acid – 3.25 min. Figure 2 shows chromatograms of a blank sample, two spiked samples and a patient sample. Blank sample responses at the retention times of 3.25 min, 3.65 min and 3.85 min. (Figure 2A) are from endogenous compounds and have peak areas below 20% of the areas of UA (3.85 min) and OA (3.65 min) peaks at the LLOQ (Figure 2A). The isomers were well separated as can be seen from the chromatograms in Figures 2B and C. The clear peaks in spiked plasma (Figure 2B) and unknown sample plasma (Figure 2D) were observed at the retention times of 3.85 min, 3.65 min, and 3.25 min for UA, OA and betulinic acid, correspondingly. Filtering samples before the LC–MS analysis and placing a C-18 ultra HPLC security guard column before the analytical column significantly reduced column clogging (data not shown). The limit of detection was determined by analyzing three samples containing 0.5 ng/mL of UA and OA and calculating signal-to-noise ratios. The limit of detection was determined to be 0.5 ng/mL with a signal-to-noise ratio of 18 utilizing this method. The peak-area ratios of standards to IS were plotted against standard concentrations to generate calibration curves and fitted with weighted linear least squares with 1/concentration weighting. For UA, the mean equation of the linear portion of the curve derived from human plasma was 0.0506x + 0.0163 (coefficient variation of slope = 6.3%), the correlation coefficient was 0.999; the linear range was from 1ng/mL to 100 ng/mL, based on a 0.5-mL sample. For OA, the mean equation of the linear portion of the curve derived from human plasma was 0.044x + 0.008 (coefficient variation of slope = 6.8%); the correlation coefficient was 0.999, and the linear range was from 1 ng/mL to 100 ng/mL, based on a 0.5-mL sample. A series of plasma controls (0.5-mL) spiked with UA and OA at 3, 30 and 80 ng/mL were analyzed for accuracy and precision (Table I). The intra-day accuracy ranged from 95% to 101%, while the inter-day accuracy ranged from 97% to 103% for both isomers. The intra-day precision varied from 4.3% to 8.8% for UA and from 2.6% to 4.1% for OA calculated from five samples in each group. The inter-day precision was 5.2–9.4% for UA and 5.9–12.2% for OA calculated from a total of 14 samples in each group (outliners which differ more than three standard deviations from the mean were removed from calculations). The standard curve concentrations were back-calculated from weighted standard curves with 1/concentration weighting (Table II). The coefficient of variation of back-calculated concentrations varied between 1.0% and 3.4% for both analytes. These results demonstrated the assay was reproducible and reliable for UA and OA quantification in human plasma. The stock solutions were stored at − 80°C and found to be stable for over a month. A stock solution was diluted with mobile phase to 10 ng/mL and analyzed before each run to test the system (Table III). Standards and controls were prepared daily in a freshly thawed plasma, human samples were stored at − 80°C and thawed once immediately before the extraction. Table I. Accuracy and precision data for ursolic acid (UA) and oleanolic acid (OA) Concentration (ng/mL) Accuracy UA (%) Accuracy OA (%) % CV UA % CV OA Intra-day (n = 5)  3 95 96 8.8 2.9  30 95 96 4.3 4.1  80 99 101 5.5 2.6 Inter-day (n = 14a)  3 98 100a 9.4 12.2a  30 96 97 5.2 5.9  80 103 103 7.0 6.5 Concentration (ng/mL) Accuracy UA (%) Accuracy OA (%) % CV UA % CV OA Intra-day (n = 5)  3 95 96 8.8 2.9  30 95 96 4.3 4.1  80 99 101 5.5 2.6 Inter-day (n = 14a)  3 98 100a 9.4 12.2a  30 96 97 5.2 5.9  80 103 103 7.0 6.5 aAn outliner which differs by three standard deviations from the mean is excluded. Table I. Accuracy and precision data for ursolic acid (UA) and oleanolic acid (OA) Concentration (ng/mL) Accuracy UA (%) Accuracy OA (%) % CV UA % CV OA Intra-day (n = 5)  3 95 96 8.8 2.9  30 95 96 4.3 4.1  80 99 101 5.5 2.6 Inter-day (n = 14a)  3 98 100a 9.4 12.2a  30 96 97 5.2 5.9  80 103 103 7.0 6.5 Concentration (ng/mL) Accuracy UA (%) Accuracy OA (%) % CV UA % CV OA Intra-day (n = 5)  3 95 96 8.8 2.9  30 95 96 4.3 4.1  80 99 101 5.5 2.6 Inter-day (n = 14a)  3 98 100a 9.4 12.2a  30 96 97 5.2 5.9  80 103 103 7.0 6.5 aAn outliner which differs by three standard deviations from the mean is excluded. Table II. Back-calculated concentrations of standards in human plasma UA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.1 10.0 20.3 50.7 101.4 SD 0.1 0.3 0.2 0.9 2.1 %CV 3.1 2.9 1.1 1.8 2.1 OA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.0 10.0 20.2 50.6 102.1 SD 0.1 0.1 0.2 1.0 1.5 %CV 3.4 1.1 1.0 1.9 1.5 UA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.1 10.0 20.3 50.7 101.4 SD 0.1 0.3 0.2 0.9 2.1 %CV 3.1 2.9 1.1 1.8 2.1 OA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.0 10.0 20.2 50.6 102.1 SD 0.1 0.1 0.2 1.0 1.5 %CV 3.4 1.1 1.0 1.9 1.5 UA, ursolic acid; OA, oleanolic acid; SD, standard deviation; CV, coefficient of variation. Table II. Back-calculated concentrations of standards in human plasma UA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.1 10.0 20.3 50.7 101.4 SD 0.1 0.3 0.2 0.9 2.1 %CV 3.1 2.9 1.1 1.8 2.1 OA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.0 10.0 20.2 50.6 102.1 SD 0.1 0.1 0.2 1.0 1.5 %CV 3.4 1.1 1.0 1.9 1.5 UA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.1 10.0 20.3 50.7 101.4 SD 0.1 0.3 0.2 0.9 2.1 %CV 3.1 2.9 1.1 1.8 2.1 OA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.0 10.0 20.2 50.6 102.1 SD 0.1 0.1 0.2 1.0 1.5 %CV 3.4 1.1 1.0 1.9 1.5 UA, ursolic acid; OA, oleanolic acid; SD, standard deviation; CV, coefficient of variation. Table III. Stock solution intensities (10 ng/mL) Validation day Ursolic acid Oleanolic acid Betullinic acid 1 76,132 62,327 86,871 2 76,155 65,513 87,533 3 72,801 57,911 83,454 Mean intensity 75,029 61,917 85,953 %SD 2.6 6.2 2.5 Validation day Ursolic acid Oleanolic acid Betullinic acid 1 76,132 62,327 86,871 2 76,155 65,513 87,533 3 72,801 57,911 83,454 Mean intensity 75,029 61,917 85,953 %SD 2.6 6.2 2.5 Table III. Stock solution intensities (10 ng/mL) Validation day Ursolic acid Oleanolic acid Betullinic acid 1 76,132 62,327 86,871 2 76,155 65,513 87,533 3 72,801 57,911 83,454 Mean intensity 75,029 61,917 85,953 %SD 2.6 6.2 2.5 Validation day Ursolic acid Oleanolic acid Betullinic acid 1 76,132 62,327 86,871 2 76,155 65,513 87,533 3 72,801 57,911 83,454 Mean intensity 75,029 61,917 85,953 %SD 2.6 6.2 2.5 The developed method was used to determine plasma concentrations of UA and OA in patients at different time points before and after consumption of apple peels as part of a clinical protocol to assess the pharmacokinetics of UA. The clinical study was approved by the University of Iowa Institutional review board (IRB# 201108728). Subjects were requested to fast overnight, eat a standardized breakfast and then consume a pre-determined quantity of apple peels as a dietary source of UA. Following ingestion of apple peels, serial blood samples were obtained, plasma separated and stored at − 80°C until analysis for the quantitation of UA. Plasma concentrations of UA (A, n = 4) and OA (B, n = 4) are shown in Figure 3. The concentration of UA was below the detection limit in all patients 30 min before the consumption. The concentration of OA was measurable in one patient and below the detection limit in three other patients 30 min before the consumption. As shown in Figure 3, the concentrations of both UA and oleanolic acid increased over time reaching a maximum concentration at ~4 h (3.3 ± 1.1 ng/mL for UA and 3.5 ± 1.5 ng/mL for OA). Concentrations of UA and OA stayed close to the maximum value for ~8 h, and then decreased to their baseline value (at 0 h) within 12–24 h. The concentrations were below the limit of detection by 48 h post-consumption in all patients. These results indicate relatively fast absorption of UA and OA and a complete clearance of isomers from the plasma in 48 h. Figure 3. View largeDownload slide Plasma concentration-time curves of ursolic (A) and oleanolic (B) acids in patients. Each point and bar represent mean ± SD (n = 4). Figure 3. View largeDownload slide Plasma concentration-time curves of ursolic (A) and oleanolic (B) acids in patients. Each point and bar represent mean ± SD (n = 4). Discussion This analytical method with a simple SLE procedure and MS detection has several advantages over other published methods. The limit of detection for ursolic and OA in this study is 0.5 ng/mL which is superior to other reported methods and avoids the process of derivatizing the sample. It allows for the rapid separation of UA and OA in plasma samples in under 5 min (total run time <7 min) with a simple extraction process. The method has been applied to both human and mouse studies, with the only modification being the sample volume, which is 0.5 mL for human studies and 0.2 mL for mouse studies. The SLE extraction utilized was easy to implement and allowed for the rapid extraction of the compounds form plasma. Filtering samples utilizing a 0.1-μm PVDF membrane (Acrodisc LC 13 mm syringe filter, Life Sciences) before the UPLC-MS analysis significantly reduced column clogging, improved the separation, and did not result in any loss of recovery (data not shown). The lower limit of detection (LLOD) values from the HPLC-MS methods in other reports (9, 13, 15) are much greater than the limit of detection observed here. Comparable LLOD values were reported for UA in rat plasma (0.5 ng/mL) and in mouse blood plasma (0.67 ng/mL) but here we show improvement without the need for derivatization and only injecting 2 μL of sample (14). The mass detector response was linear over the range of 1–100 ng/mL, a range that encompassed the plasma concentrations observed in the clinical trial or oral UA. This validated LC–MS method was developed to support the clinical development of UA for the prevention of muscle wasting and treatment of obesity. The method uses a small volume of plasma, and is sensitive enough to detect concentrations in plasma following the administration of UA to humans and can thus be applied to clinical pharmacokinetic studies in patients. Figure 3 shows a concentration time profile following the administration of UA to healthy volunteers. Plasma concentrations of UA were detected within 2 h of administration, the time to maximum concentration (Tmax) was 4 h and the elimination half-life ranged from 3.8 h to 8.2 h (mean 5.4 h). Conclusions A rapid and sensitive UPLC-MS method has been developed and validated for the determination of UA and OA in human plasma. Two isomers, UA and OA were cleaned of plasma co-eluting compounds using simple SLE method and were successfully separated using Acquity UPLC HSS column. The method is sensitive with a detection limit of 0.5 ng/mL for both analytes and has a total run time of 7 min. A small proof-of-concept study in human patients who consumed apple peels indicates that this analytical method could be used to evaluate pharmacokinetic properties of UA and/or OA in human clinical studies. Acknowledgments We would like to acknowledge the helpful discussions with Robert J. Classon at Shimadzu Scientific Instruments, Columbia, MD. Funding This work was supported by the National Institutes of Health (NIH/NIAMS 1R01AR059115-01 to C.M.A.), the Department of Veterans Affairs Biomedical Laboratory Research and Development Service (IBX000976A to C.M.A.), and grants from the Doris Duke Charitable Foundation and the Fraternal Order of Eagles Diabetes Research Center (to C.M.A.) at the University of Iowa. 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Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Rapid and Sensitive Quantification of Ursolic Acid and Oleanolic Acid in Human Plasma Using Ultra-performance Liquid Chromatography–Mass Spectrometry

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© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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Abstract

Abstract Ultra-performance liquid chromatography (UPLC) interfaced with atmospheric pressure chemical ionization mass–spectrometry was used to separate and quantify ursolic acid (UA) and oleanolic acid (OA) in human plasma. UA and OA were extracted from 0.5 mL human plasma using supported liquid extraction and separated utilizing an Acquity UPLC HSS column. The method has been validated for both UA and OA quantitation with a limit of detection of 0.5 ng/mL. The UPLC separations are carried out with isocratic elution with methanol and 5 mM ammonium acetate in water (85:15) as a mobile phase at a flow rate of 0.4 mL/min. The assay was linear from 1 ng/mL to 100 ng/mL for both analytes. The total analysis time was 7 min with the retention times of 3.25 (internal standard), 3.65 (UA) and 3.85 min (OA). Recovery of drug from plasma ranged from 70% to 115%. Analysis of quality control samples at 3, 30 and 80 ng/mL (n = 14) had an intra-day coefficient of variation of 9.9%, 4.3% and 5.5%, respectively. A proof-of-concept study in human patients who consumed apple peels indicates that this analytical method could be applied to clinical studies of UA and/or OA in human subjects. Introduction Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid; UA) and its isomer, oleanolic acid (3β-hydroxy-olea-12-en-oic acid; OA) (Figure 1) exist in natural plants and have been shown to exhibit anti-inflammatory, antifungal, antitumor and hepatoprotective pharmacological properties (1). Health-promoting effects of plants containing UA had been known for many centuries (2, 3) and recent reports of antitumor activity of the UA and OA (4) generated a wide interest in these compounds. In addition to the pharmacological properties listed above, it has recently been shown that UA reduces muscle atrophy, promotes muscle hypertrophy, and increases energy expenditure, leading to reduced obesity, improved glucose tolerance and decreased hepatic steatosis in mice (5, 6). These promising preclinical data suggest further studies in humans are warranted as UA and/or structural analogs might be useful therapeutic agents for a number of increasingly common metabolic disorders, including skeletal muscle atrophy, obesity, type 2 diabetes and non-alcoholic fatty liver disease (5, 6). Figure 1. View largeDownload slide Structures of ursolic acid, oleanolic acid and betulinic acid. Figure 1. View largeDownload slide Structures of ursolic acid, oleanolic acid and betulinic acid. UA and OA are very similar in structure, co-exist in some natural products such as apple peels, and share some common pharmacological properties. There are reports in the literature addressing isolation, separation and purification of these isomers as well as studies on their beneficial effects. The separation of these compounds still remains challenging (7). Different analytical methods have been used to separate and detect the two isomers (8–11). High performance liquid chromatography (HPLC) with ultraviolet (UV) detection (12–15), mass spectrometry (MS) (9, 16–19) or evaporative light scattering detectors (20) are the most common methods used for UA quantification in the biological samples. However, the need for a rapid and reproducible method with improved sensitivity is needed to conduct pharmacokinetic studies of UA where very low concentrations are expected. MS provides superior sensitivity compared with an UV and other detection methods, which is essential for detecting trace concentrations of UA in the plasma samples. Also, MS detection does not require extensive sample preparation, such as derivatization, and provides high resolution and rapid analysis. In this work, a method for the analysis of UA separated from OA in from plasma was developed and validated using ultra-performance liquid chromatography equipped with atmospheric pressure chemical ionization mass spectrometry (UPLC–MS). To our knowledge, this is the most sensitive and rapid method for UA detection in human plasma samples. More importantly, it also allows for the rapid separation of UA from OA, which is particularly important since both compounds are commonly found in natural products. This method has been used to support clinical studies of ursolic and OA in human subjects. Experimental Materials and reagents UA, OA, betulinic acid (Figure 1) and HPLC grade dichloromethane were purchased from Sigma-Aldrich Corp. (St Louis, MO, USA). Optima HPLC grade methanol, hexanes and American Chemical Society grade glacial acetic acid were purchased from Fisher Scientific (Pittsburgh, PA). Ammonium acetate (analytical reagent grade) was purchased from Mallinckrodt Baker Inc. (Paris, KY). HPLC grade water was from a MilliQ Integral water purification system (Millipore Corp., Bedford, MA). Human plasma was obtained from the DeGowin Blood Center at the University of Iowa Hospitals and Clinics. Stock and sample preparation To prepare plasma standards, UA and OA were dissolved separately in methanol to make 0.2 mg/mL stock solutions. About 5 μL of each solution was combined with 990 μL of methanol to form a concentrated working standard of 1 μg/mL containing both UA and OA. This standard was further diluted with methanol to 100 ng/mL to form a less concentrated working standard. Calibration standards were prepared by adding between 5 μL and 50 μL of the appropriate working standard to sufficient human plasma to prepare 0.5 mL of standards in a 2-mL Eppendorf tubes. The standard curve consisted of samples containing 1, 2, 4, 10, 20, 50 and 100 ng/mL of UA and OA. Control samples were 3, 30 and 80 ng/mL. Internal standard (IS) solution was prepared by dissolving betulinic acid to 0.2 mg/mL in methanol, and then further diluting it with methanol to 1 μg/mL. A solution containing acetic acid (4%) in water (450 μL) was added to each sample along with 10 μL of the internal standard. Blanks and controls were prepared in the same manner. All solutions were mixed and subjected to supported liquid extraction (SLE) prior to instrumental analysis. Sample extraction Isolute SLE+ 6 mL (1 mL sample volume) SLE cartridges (Part# 820-0140-C, Biotage AB, Uppsala, Sweden) were used for sample preparation. A Cerex SPE processor from Varian (Palo Alto, CA) using nitrogen gas to modulate flow was used. Samples were transferred into SLE+ cartridges and a nitrogen flow was applied for 15 s to help load the samples. The samples were allowed to adsorb onto the packing material for 10 min. The analytes were eluted with a 5-mL solvent mixture of hexanes and dichloromethane (2:1 volume:volume) under gravity flow for ~15 min. Nitrogen flow was applied to maximize sample elution for 10 s at the end. The eluates were collected in 13 mm×100 mm glass tubes and solvent was evaporated under flowing nitrogen at 25°C. The residue was reconstituted in 200 μL of 5 mM ammonium acetate in water (15%) and methanol (85%) and transferred to a 2-mL microcentrifuge tube. The samples were centrifuged at 14,000 rpm at 7°C for 30 min (Mikrocentrifuge 200 R, Hettich AG, Bäch, Swizerland). The centrifugates were filtered through a 13-mm 0.2-μm syringe filter (Life Sciences Acrodisc LC) using 1-mL plastic syringe into HPLC injection vials. Instrumentation and chromatographic conditions The instrumentation system consisted of a Waters Acquity UPLC system equipped with binary solvent manager, sample manager and column manager operating under Empower software (Waters Corporation, Milford, MA, USA) and a Shimadzu 2010A LC–MS platform in atmospheric pressure chemical ionization (APCI) negative ion mode using selected ion monitoring and operating under LC–MS Solution software (Version 2.04H3, Shimadzu, Columbia, MD, USA). An Acquity UPLC HSS analytical column, C-18, 1.8 μm, 2.1 mm × 100 mm (Waters Corporation, Milford, MA, USA) preceded by a Phenomenex C-18 ultra UHPLC security guard column was used. Separation conditions were: 7°C sample temperature; room column temperature; sample was injected in partial loop with overfill injection mode; 15 μL injection volume. Isocratic analysis was conducted at a 0.4-mL/min flow rate. Mobile phase A (15%) was 5 mM ammonium acetate; mobile phase B (85%) was methanol. The total run time for a LC–MS analysis was 7 min. The mass spectrometer was tuned using a polyethylene glycol solution following the manufacturer’s protocol. The scan interval was 0.3 s, microscan 0.1 amu, APCI temperature was 450°C, the curved desolvation line temperature (200°C), and the heat block temperature (200°C). Nitrogen flow rate was 2.5 L/min; detector voltage was 1.6 kV. The monitored mass-to-charge ratio for all three analytes was 455.45. The Waters Acquity system was configured to send a signal via a contact closure to the MS system to start data acquisition following sample injection. This allowed the Waters UPLC system to be used for sample injection and bioanalytical separation, and the Shimadzu MS to acquire the mass spectrum data even when the instruments do not normally work together in a single system. Method validation Specificity and selectivity The specificity of the method was evaluated by analyzing six different sources of blank plasma samples. Blank plasma samples were compared with spiked plasma samples at the linearity and lower limits of quantitation (LLOQ) level and the response of any co-eluting interferences calculated. Linearity and lower limits of quantitation The calibration curves were prepared by spiking blank plasma samples at seven concentrations, from 1 ng/mL to 100 ng/mL. The linearity of each calibration curve was determined by plotting the peak-area ratio of the analytes to IS versus the nominal concentration with weighted (1/concentration) least square regression. The LLOQ for the analytes were the lowest concentration with signal-to-noise ≥ 5, which could be quantitatively determined with precision and accuracy ≤ 20%, evaluated by analyzing samples in six replicates. Precision and accuracy Precision and accuracy were assessed by analyzing quality control (QC) samples in six replicates at low, medium and high concentrations of the analytes in six replicates on the same day and on three different days, respectively. Precision was defined as relative standard deviation of the measured concentration and the accuracy as the relative error of the measured mean value from the nominal value. Stability studies have previously been reported for UA and OA and are not repeated in this study (15). Results The mass spectrometric conditions were initially optimized by injecting standards at concentration of 1 μg/mL and adjusting MS voltages to improve analyte signal. The parent ion spectra for UA, OA and IS were monitored in the both positive and negative ionization with APCI and electrospray ionization (ESI) (data not shown). Due to the carboxyl in the chemical structures of UA, OA and IS, the full-scan mass spectrum showed that the signal intensity in the negative ion mode was much higher than that in the positive APCI ion mode. UA, OA and IS of endogenous compounds, are isobaric with very similar physio-chemical properties and share the same mass. Therefore, MS specificity by itself is not always adequate to separate all analytes, so we have chromatographically separated them (Figure 2). Figure 2. View largeDownload slide UPLC chromatograms of blank plasma sample (A), low standard sample (1 ng/ml) (B), middle standard sample (10 ng/ml) (C), and an unknown sample (D). Detection m/z is 455.45. Ursolic acid retention time is 3.85 min, oleanolic acid – 3.65 min., betulinic acid – 3.25 min. Figure 2. View largeDownload slide UPLC chromatograms of blank plasma sample (A), low standard sample (1 ng/ml) (B), middle standard sample (10 ng/ml) (C), and an unknown sample (D). Detection m/z is 455.45. Ursolic acid retention time is 3.85 min, oleanolic acid – 3.65 min., betulinic acid – 3.25 min. Figure 2 shows chromatograms of a blank sample, two spiked samples and a patient sample. Blank sample responses at the retention times of 3.25 min, 3.65 min and 3.85 min. (Figure 2A) are from endogenous compounds and have peak areas below 20% of the areas of UA (3.85 min) and OA (3.65 min) peaks at the LLOQ (Figure 2A). The isomers were well separated as can be seen from the chromatograms in Figures 2B and C. The clear peaks in spiked plasma (Figure 2B) and unknown sample plasma (Figure 2D) were observed at the retention times of 3.85 min, 3.65 min, and 3.25 min for UA, OA and betulinic acid, correspondingly. Filtering samples before the LC–MS analysis and placing a C-18 ultra HPLC security guard column before the analytical column significantly reduced column clogging (data not shown). The limit of detection was determined by analyzing three samples containing 0.5 ng/mL of UA and OA and calculating signal-to-noise ratios. The limit of detection was determined to be 0.5 ng/mL with a signal-to-noise ratio of 18 utilizing this method. The peak-area ratios of standards to IS were plotted against standard concentrations to generate calibration curves and fitted with weighted linear least squares with 1/concentration weighting. For UA, the mean equation of the linear portion of the curve derived from human plasma was 0.0506x + 0.0163 (coefficient variation of slope = 6.3%), the correlation coefficient was 0.999; the linear range was from 1ng/mL to 100 ng/mL, based on a 0.5-mL sample. For OA, the mean equation of the linear portion of the curve derived from human plasma was 0.044x + 0.008 (coefficient variation of slope = 6.8%); the correlation coefficient was 0.999, and the linear range was from 1 ng/mL to 100 ng/mL, based on a 0.5-mL sample. A series of plasma controls (0.5-mL) spiked with UA and OA at 3, 30 and 80 ng/mL were analyzed for accuracy and precision (Table I). The intra-day accuracy ranged from 95% to 101%, while the inter-day accuracy ranged from 97% to 103% for both isomers. The intra-day precision varied from 4.3% to 8.8% for UA and from 2.6% to 4.1% for OA calculated from five samples in each group. The inter-day precision was 5.2–9.4% for UA and 5.9–12.2% for OA calculated from a total of 14 samples in each group (outliners which differ more than three standard deviations from the mean were removed from calculations). The standard curve concentrations were back-calculated from weighted standard curves with 1/concentration weighting (Table II). The coefficient of variation of back-calculated concentrations varied between 1.0% and 3.4% for both analytes. These results demonstrated the assay was reproducible and reliable for UA and OA quantification in human plasma. The stock solutions were stored at − 80°C and found to be stable for over a month. A stock solution was diluted with mobile phase to 10 ng/mL and analyzed before each run to test the system (Table III). Standards and controls were prepared daily in a freshly thawed plasma, human samples were stored at − 80°C and thawed once immediately before the extraction. Table I. Accuracy and precision data for ursolic acid (UA) and oleanolic acid (OA) Concentration (ng/mL) Accuracy UA (%) Accuracy OA (%) % CV UA % CV OA Intra-day (n = 5)  3 95 96 8.8 2.9  30 95 96 4.3 4.1  80 99 101 5.5 2.6 Inter-day (n = 14a)  3 98 100a 9.4 12.2a  30 96 97 5.2 5.9  80 103 103 7.0 6.5 Concentration (ng/mL) Accuracy UA (%) Accuracy OA (%) % CV UA % CV OA Intra-day (n = 5)  3 95 96 8.8 2.9  30 95 96 4.3 4.1  80 99 101 5.5 2.6 Inter-day (n = 14a)  3 98 100a 9.4 12.2a  30 96 97 5.2 5.9  80 103 103 7.0 6.5 aAn outliner which differs by three standard deviations from the mean is excluded. Table I. Accuracy and precision data for ursolic acid (UA) and oleanolic acid (OA) Concentration (ng/mL) Accuracy UA (%) Accuracy OA (%) % CV UA % CV OA Intra-day (n = 5)  3 95 96 8.8 2.9  30 95 96 4.3 4.1  80 99 101 5.5 2.6 Inter-day (n = 14a)  3 98 100a 9.4 12.2a  30 96 97 5.2 5.9  80 103 103 7.0 6.5 Concentration (ng/mL) Accuracy UA (%) Accuracy OA (%) % CV UA % CV OA Intra-day (n = 5)  3 95 96 8.8 2.9  30 95 96 4.3 4.1  80 99 101 5.5 2.6 Inter-day (n = 14a)  3 98 100a 9.4 12.2a  30 96 97 5.2 5.9  80 103 103 7.0 6.5 aAn outliner which differs by three standard deviations from the mean is excluded. Table II. Back-calculated concentrations of standards in human plasma UA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.1 10.0 20.3 50.7 101.4 SD 0.1 0.3 0.2 0.9 2.1 %CV 3.1 2.9 1.1 1.8 2.1 OA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.0 10.0 20.2 50.6 102.1 SD 0.1 0.1 0.2 1.0 1.5 %CV 3.4 1.1 1.0 1.9 1.5 UA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.1 10.0 20.3 50.7 101.4 SD 0.1 0.3 0.2 0.9 2.1 %CV 3.1 2.9 1.1 1.8 2.1 OA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.0 10.0 20.2 50.6 102.1 SD 0.1 0.1 0.2 1.0 1.5 %CV 3.4 1.1 1.0 1.9 1.5 UA, ursolic acid; OA, oleanolic acid; SD, standard deviation; CV, coefficient of variation. Table II. Back-calculated concentrations of standards in human plasma UA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.1 10.0 20.3 50.7 101.4 SD 0.1 0.3 0.2 0.9 2.1 %CV 3.1 2.9 1.1 1.8 2.1 OA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.0 10.0 20.2 50.6 102.1 SD 0.1 0.1 0.2 1.0 1.5 %CV 3.4 1.1 1.0 1.9 1.5 UA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.1 10.0 20.3 50.7 101.4 SD 0.1 0.3 0.2 0.9 2.1 %CV 3.1 2.9 1.1 1.8 2.1 OA concentration (ng/mL) 4 10 20 50 100 Mean (n = 6) 4.0 10.0 20.2 50.6 102.1 SD 0.1 0.1 0.2 1.0 1.5 %CV 3.4 1.1 1.0 1.9 1.5 UA, ursolic acid; OA, oleanolic acid; SD, standard deviation; CV, coefficient of variation. Table III. Stock solution intensities (10 ng/mL) Validation day Ursolic acid Oleanolic acid Betullinic acid 1 76,132 62,327 86,871 2 76,155 65,513 87,533 3 72,801 57,911 83,454 Mean intensity 75,029 61,917 85,953 %SD 2.6 6.2 2.5 Validation day Ursolic acid Oleanolic acid Betullinic acid 1 76,132 62,327 86,871 2 76,155 65,513 87,533 3 72,801 57,911 83,454 Mean intensity 75,029 61,917 85,953 %SD 2.6 6.2 2.5 Table III. Stock solution intensities (10 ng/mL) Validation day Ursolic acid Oleanolic acid Betullinic acid 1 76,132 62,327 86,871 2 76,155 65,513 87,533 3 72,801 57,911 83,454 Mean intensity 75,029 61,917 85,953 %SD 2.6 6.2 2.5 Validation day Ursolic acid Oleanolic acid Betullinic acid 1 76,132 62,327 86,871 2 76,155 65,513 87,533 3 72,801 57,911 83,454 Mean intensity 75,029 61,917 85,953 %SD 2.6 6.2 2.5 The developed method was used to determine plasma concentrations of UA and OA in patients at different time points before and after consumption of apple peels as part of a clinical protocol to assess the pharmacokinetics of UA. The clinical study was approved by the University of Iowa Institutional review board (IRB# 201108728). Subjects were requested to fast overnight, eat a standardized breakfast and then consume a pre-determined quantity of apple peels as a dietary source of UA. Following ingestion of apple peels, serial blood samples were obtained, plasma separated and stored at − 80°C until analysis for the quantitation of UA. Plasma concentrations of UA (A, n = 4) and OA (B, n = 4) are shown in Figure 3. The concentration of UA was below the detection limit in all patients 30 min before the consumption. The concentration of OA was measurable in one patient and below the detection limit in three other patients 30 min before the consumption. As shown in Figure 3, the concentrations of both UA and oleanolic acid increased over time reaching a maximum concentration at ~4 h (3.3 ± 1.1 ng/mL for UA and 3.5 ± 1.5 ng/mL for OA). Concentrations of UA and OA stayed close to the maximum value for ~8 h, and then decreased to their baseline value (at 0 h) within 12–24 h. The concentrations were below the limit of detection by 48 h post-consumption in all patients. These results indicate relatively fast absorption of UA and OA and a complete clearance of isomers from the plasma in 48 h. Figure 3. View largeDownload slide Plasma concentration-time curves of ursolic (A) and oleanolic (B) acids in patients. Each point and bar represent mean ± SD (n = 4). Figure 3. View largeDownload slide Plasma concentration-time curves of ursolic (A) and oleanolic (B) acids in patients. Each point and bar represent mean ± SD (n = 4). Discussion This analytical method with a simple SLE procedure and MS detection has several advantages over other published methods. The limit of detection for ursolic and OA in this study is 0.5 ng/mL which is superior to other reported methods and avoids the process of derivatizing the sample. It allows for the rapid separation of UA and OA in plasma samples in under 5 min (total run time <7 min) with a simple extraction process. The method has been applied to both human and mouse studies, with the only modification being the sample volume, which is 0.5 mL for human studies and 0.2 mL for mouse studies. The SLE extraction utilized was easy to implement and allowed for the rapid extraction of the compounds form plasma. Filtering samples utilizing a 0.1-μm PVDF membrane (Acrodisc LC 13 mm syringe filter, Life Sciences) before the UPLC-MS analysis significantly reduced column clogging, improved the separation, and did not result in any loss of recovery (data not shown). The lower limit of detection (LLOD) values from the HPLC-MS methods in other reports (9, 13, 15) are much greater than the limit of detection observed here. Comparable LLOD values were reported for UA in rat plasma (0.5 ng/mL) and in mouse blood plasma (0.67 ng/mL) but here we show improvement without the need for derivatization and only injecting 2 μL of sample (14). The mass detector response was linear over the range of 1–100 ng/mL, a range that encompassed the plasma concentrations observed in the clinical trial or oral UA. This validated LC–MS method was developed to support the clinical development of UA for the prevention of muscle wasting and treatment of obesity. The method uses a small volume of plasma, and is sensitive enough to detect concentrations in plasma following the administration of UA to humans and can thus be applied to clinical pharmacokinetic studies in patients. Figure 3 shows a concentration time profile following the administration of UA to healthy volunteers. Plasma concentrations of UA were detected within 2 h of administration, the time to maximum concentration (Tmax) was 4 h and the elimination half-life ranged from 3.8 h to 8.2 h (mean 5.4 h). Conclusions A rapid and sensitive UPLC-MS method has been developed and validated for the determination of UA and OA in human plasma. Two isomers, UA and OA were cleaned of plasma co-eluting compounds using simple SLE method and were successfully separated using Acquity UPLC HSS column. The method is sensitive with a detection limit of 0.5 ng/mL for both analytes and has a total run time of 7 min. A small proof-of-concept study in human patients who consumed apple peels indicates that this analytical method could be used to evaluate pharmacokinetic properties of UA and/or OA in human clinical studies. Acknowledgments We would like to acknowledge the helpful discussions with Robert J. Classon at Shimadzu Scientific Instruments, Columbia, MD. Funding This work was supported by the National Institutes of Health (NIH/NIAMS 1R01AR059115-01 to C.M.A.), the Department of Veterans Affairs Biomedical Laboratory Research and Development Service (IBX000976A to C.M.A.), and grants from the Doris Duke Charitable Foundation and the Fraternal Order of Eagles Diabetes Research Center (to C.M.A.) at the University of Iowa. 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Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Chromatographic ScienceOxford University Press

Published: Apr 26, 2018

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