Development and Validation of an Analytical Method to Quantitate Hydroxycitric Acid, the Key Constituent in Garcinia cambogia Extract, in Rodent Plasma and Fetus

Abstract

Garcinia cambogia extract (GCE) is a popular botanical supplement used in weight loss products. Hydroxycitric acid (HCA) is the principal component in GCE. Due to lack of adequate toxicity data to assess the safe use of GCE, the National Toxicology Program is testing GCE in Hsd:Sprague Dawley® SD® rats following perinatal exposure and in adult B6C3F1/N mice. We report a validated method utilizing sample clean up with ultrafiltration followed by liquid chromatography-tandem mass spectrometry analysis to quantify HCA in rat plasma over the concentration range of 20 to 800 ng/mL. The method was linear (r² ≥ 0.99) with the limits of quantitation (LOQ) and detection (LOD) of 20.0 and 3.9 ng/mL plasma, respectively. The accuracy (determined as relative error, RE) and precision (determined as relative standard deviation, RSD) using Quality Control standards analyzed over multiple days were ≤ ± 7.5% and ≤ 9.5%, respectively. The method can be applied to quantify HCA in study matrices (RE ≤ ± 23.0%; RSD ≤ 6.0) except gestational day (GD)18 fetus. The method was partially validated in GD18 fetal homogenate over the concentration range 60-3000 ng/g (r² ≥ 0.99, RE ≤ ± 11.9%, and RSD ≤ 5.5%; LOQ 60.0 ng/g; LOD 7.77 ng/g). The standards as high as 20,000 ng/mL (plasma) and 502,000 ng/g (fetus) can successfully be quantified after diluting into the validated range (RE ≤ ± 2.6%; RSD ≤ 5.2%). These data demonstrate that the method is suitable to quantify HCA in rodent matrices and can be adapted to other biological matrices.

Introduction

Botanical dietary supplements are purported to have therapeutic effects and used worldwide by people of all ages (Wachtel-Galor and Benzie 2011). The National Institute Health’s Dietary Supplement Label Database lists over 2700 products in the botanical ingredient category (National Institues of Health 2021). Garcinia cambogia (also known as Garcinia gummi-gutta) extract (GCE) is a popular botanical ingredient used in weight loss products (Onakpoya et al. 2011; Chuah et al. 2013). It was reported as the 24^th top-selling botanical dietary supplement in mainstream outlets (e.g., grocery and drug stores) in 2019 with sales over $14 million (Smith 2020). Garcinia cambogia is also used as a traditional ingredient in Asian cuisine (Marquez et al. 2012).

Purported effects of GCE such as appetite suppression in humans via increased levels of serotonin (Ohia et al. 2001), inhibition of lipid synthesis in vivo (Peng et al. 2018), reduction of carbohydrate metabolism in human cells (Yamada et al. 2007), and fat burning via thermogenesis in humans (Campbell et al. 2016) are attributed to its principal component, hydroxycitric acid (HCA) (Figure 1). GCE is approximately 30-70% HCA by weight (Jayaprakasha and Sakariah 1998, 2000; Semwal et al. 2015). HCA was reported to be a potent inhibitor of adenosine triphosphatase citrate lyase (Hoffmann et al. 1980) in humans, and it was hypothesized that the inhibition of ATP citrate lyase results in a decreased pool of acetyl CoA available for synthesis of fat. However, studies in humans report conflicting evidence of body fat weight loss following GCE intake (Heymsfield et al. 1998; Mattes and Bormann 2000; Hayamizu et al. 2003; Roongpisuthipong et al. 2007). Some studies suggested that HCA could affect steroid hormone levels (Jena et al. 2002), however this has not been confirmed to date (Saito et al. 2005; Hayamizu et al. 2008). Potential reproductive and developmental effects of GCE have also been evaluated (Saito et al. 2005; Kiyose et al. 2006; Deshmukh et al. 2008a, 2008b; Hayamizu et al. 2008). While some studies reported no effects in rodents (Shara et al. 2003; Deshmukh et al. 2008a), other studies show toxicity to sperm (Saito et al. 2005; Kiyose et al. 2006), and a significant decrease in litter size at the high dose (10,000 ppm) (Deshmukh et al. 2008b).

Figure 1.

Structures of hydroxycitric acid, citric acid, and hydroxycitric acid lactone

Due to conflicting and inadequate data addressing the toxicity following exposure, the National Toxicology Program (NTP) is investigating the potential toxicity of GCE following exposure via feed in rats following perinatal exposure and in adult mice (NTP Testing Status). An understanding of internal exposures to HCA, including those at various stages in the developing animal, is essential to put the toxicological findings into context and extrapolate to human exposures. At the time of the initiation of the NTP program on GCE, two methods were located in the literature to quantitate HCA concentration in plasma (van Loon et al. 2000; Loe et al. 2001). These methods utilized multiple sample preparation steps, longer analysis times or had low sensitivity, suggesting that these methods were less than ideal to apply to a large number of samples potentially with a wide concentration range, as anticipated in NTP feed studies in rodents. Hence, we developed and validated a method employing an ultrafiltration step for fast and simple sample clean up followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis for sensitive and selective quantitation of HCA in rodent plasma and fetuses, and the validation results are reported herein. During reporting of this work, two additional methods to quantitate HCA in human (de Carvalho Cruz et al. 2020) and rat plasma (Bhutani et al. 2020) were identified in the literature. Both methods utilized protein participation followed by LC-MS/MS analysis.

Materials and Methods

Chemicals and Reagents

(−)-Calcium hydroxycitrate (lot No. R073A0) was procured from United States Pharmacopeia (Rockville, MD). Per manufacturer’s certificate of analysis, 0.70 mg of (−)-hydroxycitric acid is present per mg of material. This lot was used as the analytical standard to prepare the calibration and quality control (QC) samples. Citric acid-¹³C₆ (chemical purity 100%; isotopic purity 99.4%), to be used as the internal standard was received from Sigma-Aldrich (Saint Louis, MO). Amicon Ultra-0.5 centrifugal filter unit (30K MWCO, part no. UFC5030; MilliporeSigma, Burlington, MA), acetonitrile, and formic acid were purchased from Sigma-Aldrich (St. Louis, MO). Matrices to prepare calibration standards, QC samples, and matrix blanks were obtained as follows: Adult male Sprague Dawley (SD) rat plasma containing potassium ethylenediaminetetraacetic acid (K₃EDTA) as an anticoagulant was obtained from BioIVT (Westbury, NY). HSD:Sprague Dawley® SD® Rat (HSD) (adult male and postnatal day (PND) 4 male pup) plasma was purchased from BioIVT (New York, USA). PND 4 dam HSD rat plasma, and gestation day (GD)18 HSD rat fetuses were provided by Southern Research (Birmingham, Alabama). Adult male B6C3F1/N mouse plasma containing K₃EDTA as an anticoagulant was provided by Battelle (West Jefferson, Ohio).

Analytical Method Validation Approach

The analytical method was developed and validated in adult male SD rat plasma. Linearity, intraday and interday precision (determined as percent relative standard deviation (RSD)) and accuracy (determined as percent relative error (RE)) of the method were evaluated by analyzing plasma calibration standards over the HCA concentration range of 20-800 ng/mL. Quality control (QC) samples prepared in plasma (using a stock solution independent from those used for matrix calibration standard preparation) at three concentrations covering the low, medium and high end of matrix calibration range were also evaluated for intraday and interday precision and accuracy of the method. Method dilution verification was conducted to evaluate whether the concentrations above the upper limit of the validated concentration could be accurately quantitated after diluting into the validated range. Sensitivity of the method was estimated by analyzing a set of six rat plasma QC samples prepared at the lower limit of quantitation (LOQ) of 20 ng/mL for HCA. The LOQ was set based on the endogenous levels estimated in multiple blank plasma lots from plasma samples so that the LOQ would be less than 30 % of the area of the low standard and still allow for detection in the low dose samples with no interferences. The limit of detection (LOD) was calculated as three times standard deviation of the LOQ QC samples.

Absolute recovery was calculated by comparing the ratio of analyte to internal standard of spiked matrix standards to the corresponding ratio of the solvent standard. Ruggedness was tested on two chromatography columns with different serial numbers and using two analysts over the course of the validation.

Selectivity was assessed by analyzing six blanks with (method blank) and without (matrix blank) internal standard prepared in male SD rat plasma from a single lot. Analyte carryover in the instrument was evaluated using three matrix blanks (without internal standard) in the analytical run immediately following the QC sample (20,000 ng/mL). In addition, to evaluate the carryover, three solvent blanks were analyzed immediately following high matrix standard (800 ng/mL) as well as immediately following an undiluted method dilution verification QC (20,000 ng/mL) due to the presence of background HCA peaks observed in the matrices.

Reinjection reproducibility was evaluated by analyzing duplicate plasma calibration standards and four replicates of plasma QCs (15, 150, and 321 ng/mL) which were stored up to 7 days at ~2-8 °C protected from light after analysis. Samples were then re-analyzed along with stored matrix calibration standards to evaluate if they could be re-injected after storage of up to 7 days at ~ 2-8 °C.

Preparation of Solvent Calibration Standards, Plasma Calibration Standards, and Quality Control (QC) Samples

Working internal standard solution of citric acid-¹³C₆ at 125 μg/mL was prepared in filtration solvent (1% formic acid in ASTM Type 1 water). Two stock solutions of (−)-calcium hydroxycitrate were prepared in filtration solvent at 500 μg/mL with respect to HCA (corrected using 0.70 mg HCA per mg of (−)-calcium hydroxycitrate). Spiking solutions (~ 200-8000 ng/mL) were prepared from alternate stock solutions by diluting with filtration solvent. Seven matrix calibration standards were prepared over the range 20-800 ng/mL by spiking 450 μL SD male rat plasma with 50 μL of the appropriate spiking solutions. Solvent calibration standards were prepared in filtration solvent in the same range as the matrix standards. Plasma QC samples were prepared at three concentrations (30, 300, 640 ng/mL) of HCA, similar to matrix standards, using alternate stock solutions.

The samples were processed for analysis as follows: One hundred (100) μL aliquots of internal standard solution (or filtration solvent for blanks without internal standard) were transferred to individual Amicon Ultra-0.5 filters in microcentrifuge tubes followed by an aliquot (100 μL) of each calibration standard, plasma blank, or QC sample. The tubes were capped, vortexed for ~ 1 minute, and centrifuged at ~ 22,000 x g for ~ 10 minutes at ambient temperature. The filtrates were transferred to individual vials and analyzed by LC-MS/MS as described below.

Secondary Matrix Evaluation

The method validated in adult male SD rat plasma was evaluated in the following secondary matrices (e.g., study matrices): male HSD rat plasma, PND4 dam HSD rat plasma, PND4 male pup HSD plasma, male B6C3F1/N mouse plasma, and GD18 HSD rat fetus. Fetuses were homogenized prior to use. The control fetuses were thawed and the weights were recorded to the nearest 0.1 g. Water was added to fetuses (1:9 fetuses:water, w:v) and homogenized using a Ninja™ food processor. The homogenates were stored at approximately −70°C when not in use. Six replicate QC samples were prepared in each secondary matrix at 40 ng/mL (equivalent to 40 ng HCA/mL of matrix), except for GD 18 fetus where the concentration was 40 ng/mL fetal homogenate (equivalent to 400 ng (HCA)/g (fetus) based on starting fetal weight in homogenate which is one gram of fetus in every 10 mL of homogenate). Samples were quantified using the calibration curves prepared in the primary matrix (SD rat male plasma). Six matrix blanks and six method blanks were also prepared in each secondary matrix to evaluate selectivity.

Partial Validation of Fetal Homogenate

A partial validation of GD 18 fetal homogenate was conducted when the secondary matrix evaluation failed because a male SD rat plasma calibration curve did not accurately quantitate GD 18 fetal homogenate QCs. Seven calibration standards of HCA (60-3000 ng/g) and QC samples (4 replicates at 90, 900, and 2260 ng/g) were prepared in fetal homogenate to evaluate linearity, intraday precision and accuracy. Sensitivity of the method was evaluated by analyzing a set of six HSD rat fetal homogenate QC samples prepared at the lower LOQ of 60 ng/g. Method dilution verification (~ 500,000 ng/g) was conducted to evaluate whether the concentrations above the validated range could be accurately quantitated after diluting into the range. Selectivity was completed as a part of the secondary matrix evaluation above and was not repeated in the partial validation.

Stability of Hydroxycitric acid

Stability of HCA in solution (1% formic acid/water) was determined by analyzing four replicates of matrix standards prepared at the QC Mid (300 ng/mL) using HCA in 1% formic acid/water that was stored up to 47 days at ~2-8 °C compared to matrix standards prepared using freshly prepared HCA in 1% formic acid/water.

Stability of HCA in plasma during freezing and thawing of matrix was determined using four replicates each of SD rat plasma QC samples at 30 and 640 ng/mL stored at ~ −70 °C for at least 24 hours and had undergone a minimum of two freeze/thaw cycles. Stability of HCA in matrices during sample storage (~ −70 °C) was determined by analyzing four replicates each of the QC samples prepared in male HSD rat plasma at 30 and 300 ng/mL and stored for 331 and 762 days and in GD 18 fetal homogenate at 90 and 2260 ng/g stored for 63, 125, and 476 days. The stability time points were established based on the anticipated time of completion of ongoing studies and sample analysis durations. Here, we only report the data from the longest stability time points for plasma (762 days) and for fetal homogenate (476 days).

Stability of HCA in samples to cover the analysis periods and conditions (four replicates of plasma QCs at 3 levels; ~ 15, 150, and 320 ng/mL) was evaluated up to 7 days at 2-8 °C protected from light and at ambient temperature exposed to light. Samples were analyzed along with freshly prepared matrix calibration standards.

LC-MS/MS Analysis and HCA Quantitation

Samples were analyzed using a Shimadzu Prominence (Kyoto, Japan) LC coupled to AB Sciex (Framingham, MA) API 4000 or API 5000 MS in a negative ionization mode. Phenomenex Synergi Hydro-RP column (150 m x 4.6 mm (ID) x 4.0μm film thickness) (Torrance, CA) and guard column Phenomenex Security Guard (C18, 4x2 mm) were used with a flow rate of 0.5 mL/min and the mobile phases (A) 1% formic acid in ASTM Type 1 water and (B) 1% formic acid in acetonitrile. The following sawtooth gradient was used: 0.0-7.5 min, 0 to 90% B; 7.5-8.0 min, 90 to 0% B; 8.0-8.5 min, 0 to 90% B, 8.5-9.0 min, 90 to 0% B, 9.0-16.0 min 0% B. The sawtooth gradient was utilized to minimize the carry over and eliminate solvent wash injection between samples injections (Williams et al. 2012). Transitions monitored were m/z 207→217 (Supplemental Figure 1) and 197→116 for HCA and citric acid-¹³C₆, respectively. The mass spectrometer operating conditions were: ion spray voltage, −4500 V; source temperature, 550°C; Gas 1 and 2 (nitrogen), 40 psi; Collision Gas (nitrogen), 12 psi; Curtain Gas (nitrogen), 15 psi; Entrance potential, −10; Collision Energy, −20; Declustering potential, −35 and −40 for HCA and citric acid-¹³C₆, respectively; Collision Cell Exit Potential, −5 and −20 for HCA and citric acid-¹³C₆, respectively.

Calibration curves were generated by plotting the peak area ratios of analyte to internal standard in plasma and fetal homogenate as a function of analyte concentration. A linear regression with 1/x weighting was used to relate peak area response ratio of analyte to internal standard and concentration of HCA in corresponding matrix. Plasma concentration of HCA were reported as ng/mL. Fetal homogenate concentration in ng/mL homogenate was converted to ng/g fetus, after adjusting for homogenate volume.

Results and Discussion

Analytical Method Development

The NTP is investigating potential toxicity of GCE following exposure in rodents to a wide range of concentrations (0-30000 ppm) via feed. Assessing internal exposures, especially during critical stages in the development (i.e., gestation and lactation) of the model animal, is essential to contextualizing the toxicological findings and translating findings to human exposure scenarios. At the initiation of this project, there were two methods in the literature to quantify HCA in rodent and human plasma (van Loon et al. 2000; Loe et al. 2001) and no methods were available for tissues, such as fetuses. These existing methods used either a non-specific ion chromatography method (van Loon et al. 2000) or a gas chromatography-mass spectrometry method requiring derivatization (Loe et al. 2001) for HCA quantitation. In addition, multi-step sample preparation and clean-up steps, such as protein precipitation followed by titration and salt precipitation, were required. The challenges with existing methods led us to undertake this work to develop a fast and efficient method to quantitate large numbers of samples, potentially with a wide range of concentrations to characterize internal exposure to HCA. During a recent survey of the literature, while preparing the data for reporting our work, we identified two additional methods to quantitate HCA in plasma, both utilizing protein precipitation followed by LC-MS/MS analysis which have been successfully applied to pharmacokinetic studies of HCA (Bhutani et al. 2020; de Carvalho Cruz et al. 2020). The authors didn’t report the application of the methods to other matrices.

It has been reported that HCA in solvent converts to the less reactive lactone over time (Soni et al. 2004; Venkateswara Rao et al. 2010; Bakhiya et al. 2017) (Figure 1). This combined with the poor solubility of HCA in organic solvents (LogP −2.6) (PubChem 2021) led us to utilize size exclusion with Amicon ultra centrifugal filter units and 1% formic acid in ASTM Type 1 water for sample clean up instead of protein precipitation using organic solvents. In addition, these filters have been shown to provide fast and simple sample processing and promote high sample recoveries (MilliporeSigma 2021). Hence, we utilized these ultrafiltration filters for sample clean up followed by analysis using LC-MS/MS. Because isotopically labelled HCA was not available commercially, we evaluated several commercially available isotopically-labeled acids (citric-2,2,4,4-d₄ acid, 1,8-octanedioic-d₁₂ acid, and citric acid-¹³C₆) as potential internal standards. Citric acid-¹³C₆ (Figure 1) was selected due to better reproducibility throughout the method development (data not shown).

The method was successfully validated to quantify HCA in rodent plasma and fetal homogenate. Endogenous HCA was present in blank matrices and found concentrations are reported in Table 1. An average concentration of ~ 5 ng/mL was estimated in blank SD rat plasma and 1.0 ng/mL (~ 10 ng/g) in fetal homogenate. Hence, LOQs of 20 ng/mL or 60 ng/g were selected for the validation in plasma and fetal homogenate, respectively, so the endogenous level would be less than 30% of the area of the lowest standard of the calibration curve (LOQ). Representative chromatograms for HCA in control SD rat plasma and HSD fetal homogenate are presented in Figures 2 and 3, respectively. To the best of our knowledge the endogenous HCA levels have not previously been reported in rodents.

Table 1.

Endogenous hydroxycitric acid concentration (HCA) in Sprague Dawley (SD) rat, Hsd:Sprague Dawley® SD® (HSD) rat and B6C3F1/N mouse matrix blanks

Species/Matrixa	Endogenous HCA (%)b (Mean ± SE)	Endogenous HCA concentration (ng/mL, ng/g)c (Mean ± SE)
SD Rat Plasma	25.4±1.00	5.25±0.21
Male HSD Rat Plasma	29.6±1.28	6.11±0.26
PND4 Dam HSD Rat Plasma	35.1±3.77	7.25±0.78
PND4 Male HSD Rat Pup Plasma	26.4±0.93	5.44±0.19
Male B6C3F1/N Mouse Plasma	35.8±1.95	7.38±0.40
GD18 HSD Rat Fetus Homogenate	16.3±2.69	10.1±1.71

^aTotal of 12 sample; n=6 blank matrix and n=6 blank matrix + internal standard per matrix

^bExpressed as a % of the LOQ, The LOQ for HCA is approximately 20 ng/mL for plasma and 60 ng/g for fetal homogenate.

^cExpressed as an extrapolated concentration as ng/mL and ng/g for plasma and fetal homogenate, respectively.

Figure 2.

Representative LC-MS/MS chromatogram of hydroxycitric acid for blank Sprague Dawley (SD) plasma with and without internal standard (IS), low and high hydroxycitric acid in SD rat plasma; A1: Full scale, A2: Reduced scale

Figure 3.

Representative LC-MS/MS chromatogram of hydroxycitric acid for HSD:Sprague Dawley® SD® (HSD) rat fetal homogenate with and without internal standard (IS), low and high hydroxycitric acid in HSD in fetus; A1: Full scale, A2: Reduced scale

Analytical Method Validation

The analytical method was fully validated in male SD rat plasma (primary matrix) and data are presented in Table 2. The plasma calibration curves were linear with coefficients of determination (r²) values > 0.997 (Supplemental Figures 2 and 3).

Table 2.

Method validation data for hydroxycitric acid (HCA) in male Sprague Dawley (SD) rat plasma

Matrix Concentration Range (ng/mL)	20-800
Coefficient of Determination (r2)	> 0.997
LOD a (ng/mL)	3.9
LOQ (ng/mL)	20
Accuracy (RE % b)
Intra-Day c	≤ ±7.2
Inter-Day c	≤ ±5.6
Intra-Day d	≤ ±7.5
Inter-Day d	≤ ±6.2
Precision RSDe (%)
Intra-Day c	≤ 9.6
Inter-Day c	≤ 5.6
Intra-Day d	≤ 9.5
Inter-Day d	≤ 5.8
Method Dilution Verification f
Accuracy (RE %)	0.0
Precision (RSD %)	5.2
Reinjection Reproducibility
Accuracy (RE %)	≤ 4.7
Precision (RSD %)	≤ 5.7
Absolute Recovery % g	130.0 to 192.5

^aLOD = Limit of Detection; estimated as 3 x standard deviation of the lower limit of quantitation; the lowest concentration of the calibration curve (LOQ) (n=6)

^b%RE = percent relative error

^cEstimated based on plasma calibration standards

^dEstimated based on low, mid and high-level (30, 300, and 640 ng/mL, respectively) Quality Control (QC) samples n = 4 for intra-day and n = 12 for inter-day

^e%RSD = percent relative deviation

^fEstimated based on matrix (SD rat plasma) QC samples QC at 20000 ng/mL; diluted 40-fold with SD rat plasm prior to processing, n = 4

^gAbsolute recovery samples were prepared for HCA (n=2/concentration, 7 concentrations) over the concentration range of 20-800 ng/mL

The intra- and inter-day accuracy (determined as % RE) were ≤ ±7.2 and ≤ ±5.6, respectively, for plasma calibration standards. Intra- and inter-day precision (determined as % RSD) were ≤ 9.6 and 5.6, respectively (Table 2) demonstrating the good performance of the calibration curves within and between multiple days.

Assay performance was assessed using plasma QC samples. Intra- and inter-day % RE were ≤ ±7.5% and ±6.2%, respectively. Intra- and inter-day RSD were ≤ 9.5% and 5.8%, respectively (Table 2). These values are within those recommended by the Food and Drug Administration for bioanalytical method validation (The U.S. FDA 2018). For similar methods (Bhutani et al. 2020; de Carvalho Cruz et al. 2020), intra- and inter-day % RE and RSD were reported as ≤ ±10.0 and ≤ 12.0, respectively.

The performance of the assay at the LOQ was determined by analyzing six plasma QC samples prepared at 20 ng/mL. This concentration was selected based on the anticipated endogenous concentrations in rodent plasma as described above (e.g., endogenous concentrations would be less than 30% of the area of the LOQ). The determined RE value at LOQ was −0.5% and RSD was 6.3%. The LOD, estimated as three times standard deviation of LOQ, was 3.90 ng/mL. The LOQs reported previously in rat and human plasma (10.5 and 50 ng/mL, respectively) were similar to the ones determined in our study (Bhutani et al. 2020; de Carvalho Cruz et al. 2020). A LOD value of 15 ng/mL was reported in human plasma (de Carvalho Cruz et al. 2020).

Selectivity samples from one lot were processed without internal standard and evaluated for the response of any peak with the same retention times as the HCA or internal standard with >30% of the average response of the LOQ standards. Based on this criteria, there were no interfering peaks at the approximate retention time of HCA or internal standard in 10 of the 12 replicates analyzed. Two replicates had peak areas slightly greater than 30% (30.1 and 32.9%).

To evaluate the dilution of samples with concentrations above the upper limit of quantitation, rat plasma method dilution verification QC samples, ~ 20,000 ng HCA/mL (n=4) were prepared. Results demonstrated that these samples could be diluted with plasma into the validated concentration range with a RE of 0.0% and RSD of 5.2% (Table 2).

Overall assay recovery (absolute recovery) of analyte which includes the ultrafiltration was evaluated using calibration standards at all concentrations by comparing the ratio of analyte to internal standard of spiked matrix standards to the corresponding ratio of the solvent standards. The estimated values were higher than 100% (130-192.5%) (Table 2), suggesting a matrix enhancement and the quantitation of HCA in matrix samples requires the use of respective matrix curve. This can be eliminated by use of an isotopically labelled HCA as an internal standard which behaves similarly as the analyte in the mass spectrometer. However, isotopically labeled HCA is not commercially available. Since matrix curves prepared in respective matrices were used for the quantitation of HCA in plasma and fetal homogenate samples, observed high absolute recoveries doesn’t affect the quantitation of HCA in this study as has been demonstrated with QC samples where RE ≤ ± 7.5% and RSD ≤ 9.5% (Table 2) during. The absolute recovery reported in the literature using similar methods was ~ 98% (Loe et al. 2001) or ~ 85% (Bhutani et al. 2020).

Carryover for HCA or internal standard was evaluated by injecting a rat plasma blank immediately following a method dilution verification QC injection (20,000 ng/mL). The area response in the blank sample when compared to that of the LOQ was ≤ 30% for HCA. The value was consistent for all three injections indicating this was endogenous HCA and not carryover. To further evaluate the carryover, three solvent blanks were analyzed immediately following a high matrix standard (800 ng/mL) and immediately following an undiluted method dilution verification QC (20,000 ng/mL). The area response in the blank sample when compared to that of the LOQ was 0% for HCA demonstrating no carry over and that what was seen in the plasma blank was due to endogenous HCA and not carry over.

Reinjection reproducibility had average determined concentrations of the QC samples with RE of ≤ ±4.7% of nominal and RSD values ≤ 8.7%, at each concentration level upon reinjection (Table 2). The results indicate that an entire set of rat plasma samples, including standards and QCs prepared on the same day as the samples, can be reinjected when stored at the temperature of the autosampler (refrigerated) for up to 7 days. Batches of approximately 70 rat validation sample injections conducted by two different analysts and using two different LC columns were accurate with RE ≤ ±15% of nominal and precise with RSD ≤ 15% at each concentration level demonstrating the ruggedness of the method (data not shown).

Secondary Matrix Evaluation

The method was evaluated in the following anticipated study matrices: adult male HSD rat plasma, PND 4 dam HSD rat plasma, PND 4 pup male HSD rat plasma, GD18 HSD rat fetal homogenate, and adult male B6C3F1/N mouse plasma. QC samples were prepared in respective matrices and were quantified using the calibration curves prepared in the primary matrix (SD rat male plasma). The RE was 3.9% to 23.0% and RSD was ≤ 6.0% demonstrating the suitability of the assay to quantitate HCA in study matrices (Table 3), except for GD18 fetal homogenate. In fetal homogenate the RE was −84.8 to −81.9%, indicating that a fetal homogenate matrix curve should be utilized instead of plasma curves for the analysis of HCA in fetus samples. The secondary matrix blanks from three different lots, in general, had no interfering peaks (peak area greater than 30% of the LOQ, 20 ng/mL, for HCA), with exception of 2 lots for PND4 dam HSD rat plasma and 2 lots for male B6C3F1/N mouse plasma. The lack of interfering peaks demonstrated that the method was selective in all the secondary matrices. No literature methods were identified for the analysis of HCA in male B6C3F1/N mouse plasma, male HSD rat plasma, PND 4 dam HSD rat plasma, or PND 4 pup plasma.

Table 3.

Secondary matrix evaluation for hydroxycitric acid (HCA) in Hsd:Sprague Dawley® SD® (HSD) rat plasma, male B6C3F1/N mouse plasma and GD18 HSD rat fetal homogenate

Species/Matrixa	HCA
Male HSD Rat Plasma
Accuracy (RE %)	≤ ±17.5
Precision (RSD %)	≤ 4.6
PND4 Dam HSD Rat Plasma
Accuracy (RE %)	≤ ±20.1
Precision (RSD %)	≤ 2.8
PND4 Male Pup HSD Rat Plasma
Accuracy (RE %)	≤ ±23.0
Precision (RSD %)	≤ 2.4
Male B6C3F1/N Mouse Plasma
Accuracy (RE %)	≤ ±11.2
Precision (RSD %)	≤ 6.0
GD18 HSD Fetal Homogenate b
Accuracy (RE %)	≤ ±84.8
Precision (RSD %)	≤ 6.0

^aSix replicates matrix QCs at 200% of lower limit of quantitation (LOQ) (~40 ng/mL); RE %= percent relative error; RSD % = percent relative deviation.

^bSix replicates matrix QCs at 200% of LOQ (~40 ng/mL equivalent to ~400 ng/g (based on tissue weight, 1 g of tissues in 10 mL of homogenate)) Failed, triggered the partial validation

Partial Validation in Fetal Homogenate

QC samples prepared in GD18 rat fetal homogenate, when quantified using a plasma calibration curve, were precise (RSD:6.0%), but gave poor accuracy (%RE: −84.8 to −81.9%) due to matrix differences between the plasma and fetal homogenate. Therefore, a partial validation was performed using fetal homogenate matrix standards over a calibration range of 60 to 3000 ng/g (Table 4). The method was linear using a 1/x weighting factor with r² = 0.993 (Supplemental Figure 4). Intra-day RE were ≤ ±12.3% and 11.9% and RSD were ≤ 14.5% and 5.5% for all calibration standards and QC samples, respectively (Table 4). The LOQ was approximately 60 ng/g with RE and RSD values of 4.9% and 4.1%, respectively. The LOD in GD18 HSD rat fetus homogenate was 7.77 ng/g. Method dilution verification, which was conducted at ~500,000 ng/g demonstrated that samples could be successfully diluted into the validation range with RSD values of ≤ 2.8%, and an average % RE of 2.6%. To the best of our knowledge there are no methods available in the literature to quantitate HCA in tissues including fetus.

Table 4.

Partial validation of hydroxycitric acid (HCA) in GD18 Hsd:Sprague Dawley® SD® rat fetal homogenate

Matrix Concentration Range (ng/g)	60-3000
Coefficient of Determination (r2)	> 0.993
LOD a (ng/g)	7.77
LOQ (ng/g)	60.0
Accuracy (RE b%)
Intra-Day c	≤ ±12.3
Intra-Day d	≤ ±11.9
Precision RSDe (%)
Intra-Day c	≤ 14.5
Intra-Day d	≤ 5.5
Method Verification
Accuracy (REb %)	2.6
Precision (RSDe %)	2.8

^aLOD = Limit of Detection; 3 x Standard Deviation for 6 lower limit of quantitation (LOQ) replicates

^b%RE = percent relative error

^cEstimated based on fetal homogenate calibration standards

^dEstimated based on fetal homogenate on low-, mid-, and high-level (90, 900, and 2260 ng/g, respectively) Quality Control (QC) samples

^e%RSD = percent relative deviation.

Stability

The stability data for the primary matrix (SD rat plasma) is presented in Table 5. The stability results for HCA showed RE ≤4.4% and RSD of ≤8.7%. The data demonstrated that the filtered plasma samples are stable and hence could be reinjected when stored at the temperature of the autosampler (refrigerated) for up to 6 days.

Table 5.

Stability data for hydroxycitric acid (HCA) in male Sprague (SD) rat plasma, male Hsd:Sprague Dawley® SD® (HSD) rat plasma, GD18 HSD fetal homogenate, and in solution (1% formic acid/ASTM type 1 water)

Stability (RE %)	HCA
Freeze-Thaw SD Rat Plasma a	105.6 to 107.1 % Day 0
SD Rat Plasma (−70 °C) b	89.2 to 102.4 % Day 0
GD18 HSD Fetal Homogenate (−70 °C) c	92.3 to 103.2 % Day 0
Filtrate Stability, Refrigerated in Dark d	95.6 to 102.6 % Day 0
Filtrate Stability, Ambient in Light e	88.3 to 96.0 % Day 0
Analyte Solution Stability, Refrigerated in Dark f	≤±4.9 %REg
Internal Standard Solution Stability, Refrigerated in Dark h	≤±9.0 %REg

^aFour replicates matrix QCs at 2 levels (30 and 640 ng/mL); underwent two freeze and thaw cycles (~−70°C/ambient temperature)

^bFour replicates matrix QCs at 2 levels (30 and 300 ng/mL); stored at ~ −70°C for 762 days

^cFour replicates matrix QCs at 2 levels (90 and 2260 ng/g); stored at ~ −70°C for 476 days

^dFour replicates plasma QCs at 3 levels (15, 150, and 321 ng/mL); filtered, stored refrigerated in dark for 6 days, and injected with freshly prepared matrix calibration standards

^eFour replicates plasma QCs at 3 levels (15, 150, and 321 ng/mL); filtered, stored at ambient temperature in the light for 4 days, and injected with freshly prepared matrix calibration standards

^fFour replicates of a matrix QC sample at ~300 ng/mL prepared using HCA in 1% formic acid/water at ~ 500 μg/mL; stored refrigerated (~ 2-8°C) for 47 days

^g%RE = percent relative error

^hFour replicates of a matrix QC sample at ~ 300 ng/mL prepared using citric acid-¹³C_{6 in} 1% formic acid/water at 125 ng/mL; stored refrigerated (~ 2-8°C) for 47 days

Stability of the HCA and internal standard in 1% aqueous formic acid was evaluated by storing solutions at 2-8°C protected from light for a minimum of 47 days, then using the stored solutions to prepare four aliquots of plasma samples prepared at the mid QC concentrations (300 ng/mL) and analyzing them. Analytes were found to be stable up to 47 days for both HCA and internal standard with measured concentrations ≥ 90% of the nominal concentrations (RSD ≤1.6 and ≤3.4 %, respectively).

Freeze-thaw plasma (matrix) storage stability was performed through two freeze-thaw cycles (−70°C/ambient temperature) protected from light. The recovery compared to Day 0 and RSD for HCA were 105.6% (low QC), 107.1% (high QC), and ≤ 2.7%, respectively.

Plasma storage stability was performed for up to 762 days of storage at ~ −70°C. The recovery relative to Day 0 was 96.6% (low QC) and 108.9% (high QC) with RSD values ≤ 2.7%. Stability of HCA in rodent or human plasma when stored at (~ −20 °C) for up to 186 days has been demonstrated by other investigators (Bhutani et al. 2020; de Carvalho Cruz et al. 2020).

Fetal homogenate storage stability was performed for up to 476 days at ~ −70°C. The recovery relative to Day 0 concentration was 92.3% (low QC) and 103.2% (high QC) with RSD values of ≤3.5%. No literature data was found for stability of HCA in fetal homogenate.

Conclusions

An LC-MS/MS method was validated to quantitate HCA in SD rat plasma and fetal homogenate. The quantitation ranges were 20-800 ng/mL in plasma and 60-3000 ng/g in fetal homogenate, using 100 μL aliquots of plasma or fetal homogenate. LODs were 3.9 ng/mL and 7.77 ng/g for plasma and fetal homogenate, respectively. REs were ≤ ± 7.5% and ≤ 12.3%, and RSDs were ≤ 9.6% and ≤ 14.5% for plasma and fetal homogenate, respectively, indicating that the method was accurate and precise. HCA in rat plasma and fetal homogenate was found to be stable when stored ~ −70 °C for up to 762 and 476 days, respectively. Both primary matrix and secondary matrices were selective for HCA and internal standard. Samples above the upper limits of quantitation could be successfully diluted into the range. These data show that the method is suitable for determination of HCA in plasma and fetuses from toxicology studies. In addition, this method could be easily adapted to other biological matrices.