Simultaneous quantification of free curcuminoids and their metabolites in equine plasma by LC-ESI-MS/MS

Journal of Pharmaceutical and Biomedical Analysis 154 (2018) 31–39

Simultaneous quantification of free curcuminoids and their metabolites in equine plasma by LC-ESI-MS/MS

Yongqiang Liu a, Melissa Siard b, Amanda Adams b, Michael L. Keowen c, Travis K. Miller c,Frank Garza, Jr. c, Frank M. Andrews c, Navindra P. Seeram a,*

  1. Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, 7Greenhouse Road, Kingston, RI, 02881, USA
  2. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, 1400 Nicholasville Road, Lexington, KY, 40546, USA
  3. Equine Health Studies Program, Department of Veterinary Clinical Sciences, Louisiana State University School of Veterinary Medicine, Skip Bertman Drive,1843 Veterinary Teaching Hospital, Baton Rouge, LA, 70803, USA

∗ Corresponding author.
E-mail address: nseeram@uri.edu (N.P. Seeram).

Article Info

Article history:

  • Received 12 January 2018
  • Received in revised form 5 March 2018
  • Accepted 6 March 2018

Keywords:

  • Curcuminoids
  • Metabolites
  • Equine
  • Plasma
  • LC-MS/MS
  • Pharmacokinetics

Abstract

The human health benefits attributed to turmeric/curcumin spice has resulted in its wide utilization as a dietary supplement for companion pets and other animals including horses. While the quantification of free curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin) and their phase-2 metabolites (curcumin-O-sulfate, curcumin-O-glucuronide) have been extensively investigated in human and rodent biological samples (primarily plasma and serum), there is lack of similar data for horses. Herein, we report a validated LC-ESI–MS/MS method for the simultaneous quantification of the afore-mentioned free curcuminoids and their metabolites in equine plasma. The linearity of the aforementioned curcuminoids and curcumin-O-sulfate was in the range of 0.5–1000 ng/mL and 1–1000 ng/mL for curcumin-O-glucuronide with 85–115% accuracy and <15% precision in equine plasma. The method was validated based on US FDA criteria and applied to characterize the pharmacokinetics of curcumin-O-sulfate in equine plasma.

© 2018 Elsevier B.V. All rights reserved.

1. Introduction

Turmeric (also commonly referred to as curcumin food spice), which is the rhizome of the Curcuma longa L. plant, has been extensively studied for a wide range of biological effects [1–4]. Natural turmeric curcumin consists of a mixture of three ‘free’ curcuminoids, namely, curcumin (1), demethoxycurcumin (2), and bisdemethoxycurcumin (3). Because curcumin (1) is the major bioactive compound in turmeric curcumin, it’s the most widely studied of the three curcuminoids. However, the poor water solubility and oral bioavailability of free curcumin (1) has been regarded as the most significant limitation for its use as a therapeutic agent [5–7]. After oral consumption, curcumin (1) undergoes phase-2 metabolism to form two major metabolites, curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) [8].

The anti-inflammatory and other biological effects attributed to turmeric curcumin has led to its utilization in pet food formulations and dietary supplements for animals, including small mammals such as dogs and cats, as well as horses. Several published methods using sensitive analytical methods, namely, liquid chromatography mass spectrometry (LC–MS), are available for the quantification of free curcuminoids and their metabolites in biological samples (primarily, plasma and serum) of rodents (rat and mice) [9–12] and humans [13–15]. However, to date, the quantification of these compounds in equine plasma has not been reported.

Given the well-established biological effects of curcumin [1–4], our laboratory has designed future studies to evaluate the anti-inflammatory effects of a curcumin formulation (Longvida®), which has previously been studied in rats, mice, and human subjects [7–19] and in vitro studies in horses [20,21]. In preparation for the analyses of biological samples collected from horses, and given that there are no published validated methods for the quantification of curcumin and its metabolites in equine plasma, we designed the current study. Herein, we report the development of a validated LC-ESI–MS/MS method for the simultaneous quantification of the aforementioned free curcuminoids and metabolites in equine plasma. The method was validated based on US FDA criteria and applied to characterize the pharmacokinetics of curcumin-O-sulfate in equine plasma after an oral dose of a supplement containing Longvida® in two horses. The chemical structures of the curcuminoids curcumin (1), demethoxycurcumin (2), and bisdemethoxycurcumin (3), the phase-2 metabolites of curcumin, curcumin-O-sulfate (4), and curcumin-O-glucuronide, (5) and the internal standard, urolithin B (6) are shown in Fig. 1. This is the first reported US FDA validated method for the quantification of these compounds in equine plasma.

Fig. 1 Chemical structures of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and the internal standard urolithin B (6).

2. Materials and methods

2.1. Reagents and chemicals

LC–MS grades of acetonitrile and formic acid, demethoxycurcumin (Batch: 0000014666 and bisdemethoxycurcumin (Lot: 096M4730V) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Curcumin (batch: HWI01648-3) was purchased from HWI Pharma Services GmbH (Rheinzabern, Germany). Curcumin-O-sulfate (Lot: 1617-009A2) and curcumin-O-glucuronide (Lot: 2681-095A5) were purchased from TLC PharmaChem (Mississauga, Ontario, Canada). Urolithin B (6) was synthesized as previously reported by our laboratory [22]. All the standards with purity of >97% by HPLC. Dimethylsulfoxide (DMSO) was obtained from Pharmco-AAPER through Wilkem Scientific (Pawcatuck, RI, USA). Blank equine plasma from 6 horses were provided by the Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky.

2.2. Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS)

Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS) analyses were performed on a Prominence UFLC system (Shimadzu, Marlborough, MA, USA) coupled to a QTRAP 4500 system (Applied Biosystems/MDS Sciex, Framingham, MA, USA) with data acquired using Analyst 1.6.3 software and processed using MultiQuant 3.0.1 software. The UFLC system consisted of three LC-20AD pumps, a DGU-20A degassing unit, SIL-20AC auto sampler, CTO-20AC column oven and CBM-20A communication bus module. Chromatographic separation was performed on a 100 mm x 2.1 mm i.d., 2.5μm, XBridge BEH C18 column Waters, (Milford, MA, USA) with mobile phase A [acetonitrile containing 0.1% (v/v) formic acid] and mobile phase B [water containing 0.1% (v/v) formic acid]. Elution was completed using a 9-min isocratic program (43% A; 57% B) operating with a 0.2 mL/min flow rate with an 8μL injection volume. The column temperature was maintained at 40°C. The MS operated in electrospray ionization (ESI) in negative mode with multiple reaction monitoring (MRM). Nitrogen was used as the source gas in all cases. Parameters were optimized as follows: IonSpray voltage, -4500 V; nebulizer gas, 35; auxiliary heater gas, 45; curtain gas, 25; turbo gas temperature, 300°C. Table 1 shows a summary of the optimized mass spectrometry parameters.

2.3. Preparation of equine plasma solutions

2.3.1 Preparation of calibration standards and internal standard solutions

The primary stock solutions of the standards, curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) were prepared by dissolving each compound in 0.2 mL DMSO and diluting with acetonitrile to achieve final concentrations of 1 mg/mL. A stock solution of the internal standard, urolithin B (6) was prepared by dissolving in acetonitrile to achieve a final concentration of 1 mg/mL. All samples were stored at -20°C. To prepare calibration curves, an aliquot of 10 μL of the appropriate diluted standard solution (a mixture of curcumin (1), demethoxycurcumin 2, bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5), each at 5–10,000 ng/mL, and the internal standard, urolithin B (6), at 10,000 ng/mL) were spiked into an aliquot of equine plasma to yield samples containing 0.5–1000 ng/mL standards and 1000 ng/mL of the internal standard, urolithin B (6), respectively. The samples were vortexed for 10 s. Then, samples were vortexed for 30 s after which 200 μL acetonitrile was added for protein precipitation. After allowing the samples to sit at 4°C for 10 min, the mixtures were centrifuged for 10 min. The supernatant was collected and diluted with 100 μL DI water containing 0.5% (v/v) formic acid then subjected to LC-ESI–MS/MS analysis.

Table 1
Optimized mass spectrometry conditions for the five analytes and the internal standard, urolithin B (6)

2.3.2. Preparation of quality control (QC) solutions

Quality control (QC) samples were prepared in equine plasma for curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5), each at 10, 100, and 500 ng/mL. The samples were vortexed for 10 s. Then, samples were vortexed for 30 s after which 200μL acetonitrile was added for protein precipitation. After allowing the samples to sit at 4°C for 10 min, the mixtures were centrifuged for 10 min. The supernatant was collected and diluted with 100μL DI water containing 0.5% (v/v) formic acid then subjected to LC-ESI–MS/MS analysis.

Working solutions for calibration samples and QC samples were prepared fresh every week. Calibration standards were prepared fresh daily for analyses during the validation.

2.4. LC-ESI–MS/MS method validation

The developed LC-ESI–MS/MS method was validated according to the USA FDA guidelines [23] for specificity, accuracy, precision, recovery, matrix effect, and stability as described below.

2.4.1. Specificity

The specificity of the method was evaluated by analyzing blank equine plasma and blank equine plasma spiked with the five analytes, curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) (each at 50 ng/mL) and the internal standard, urolithin B (6) (at 1000 ng/mL).

2.4.2. Calibration curves

The calibration curves were acquired by plotting the ratio of peak area of analytes to that of the internal standard against the nominal concentration of the calibration standards. The results were fitted to linear regression analysis using 1/X2 as weighting factor.

2.4.3. Accuracy and precision

The intra-day validation was determined in six replicates each of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3) and curcumin-O-sulfate (4) each at 1, 10, 100, and 500 ng/mL, and curcumin-O-glucuronide (5) (2, 10, 100 and 500 ng/mL). The interday validation was determined across these concentrations in triplicates on three different days. The mean concentrations and the coefficient of variation (CV) of intra-day were calculated as the relative standard deviation (%) from the replicates the (CV) of inter-day was calculated as the relative standard derivation (%) of the respective mean concentrations on each individual day for three days. The accuracy of the assay was determined by comparing the corresponding calculated mean concentrations with the nominal concentrations.

2.4.4. Recovery and matrix effect

A post-extraction spike experiment was performed to evaluate the recovery and matrix effects of curcumin (1), demethoxycur-cumin 2, bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and, the internal standard, urolithin B (6), in equine plasma. Recovery and matrix effects were evaluated in five replicates at three different QC concentrations. Three separate batches of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) each at 10, 100, and 500 ng/mL, and the internal standard, urolithin B (6) (1000 ng/mL), were prepared as follows: (batch A) curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and the internal standard, urolithin B (6) were prepared in 43% aqueous acetonitrile containing 0.1% (v/v) formic acid; (batch B) 100μL plasma was vortexed for 30 s after which 200μL acetonitrile added for protein precipitation. After allowing to sit at 4°C for 10 min, the mixtures were centrifuged for 10 min. The supernatant was collected and diluted with 100μL DI water containing 0.5% (v/v) formic acid, then curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and the internal standard, urolithin B (6) were spiked into the solution batch C curcumin (1), demethoxycurcumin 2, bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and the internal standard, urolithin B (6) were treated in the same method as for the QC samples. The recovery of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) was calculated by the ratios of the corresponding peak areas of the samples in batch C to those of the samples in batch B. The matrix effect was evaluated by the ratio of the peak areas of the samples in batch B to those of the samples in batch A.

2.4.5. Stability

The stability of the analytes was assessed after storage at -80°C for 15 days. Briefly, standard stock solutions were added to each equine plasma to yield curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) each at 10, 100 and 500 ng/mL, on days 1, 8, and 15. The samples were immediately stored at 80°C. On day 16, all of the samples were analyzed using the LC-ESI–MS/MS method. To evaluate freeze-thaw stability, freshly prepared equine plasma samples, all at the above concentrations, were exposed to three sequential freeze-thaw cycles prior to sample analyses. During each cycle, the samples were thawed at 4°C for approximately 3 h and stored at -80°C for the next freezing cycle at 24 h intervals. To evaluate the post-extraction stability in the solution in the auto-sampler 4°C, samples at concentrations of 10, 100 and 500 ng/mL were subjected to LC-ESI–MS/MS analyses at 0, 3, 6 and 9 h. The accuracy was calculated as the % relative error (RE).

2.5. Analysis of curcumin (1), demethoxycurcumin (2),bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) in equine plasma samples

Our group has a planned study (at Louisiana State University) designed to evaluate the effects of an oral herbal supplement 2 oz containing several ingredients including a curcumin formulation (Longvida®; 1.06 g) for easing pain and improving lameness in horses due to osteoarthritis. Treatment consisted of 2 ounces of the supplement for 30 days and blood samples were obtained from two horses (Equine 1=580.6 kg; Equine II=411.4 kg) on days 1 and 14 to determine plasma concentrations of aforementioned curcuminoids metabolites. Blood samples were collected at 0, 15, 30, 45, 60, 75, 90, 105, 120, 150, 180, 210, 240, 270, 300, 330, 360 min, then 7, 8, 9, h in tubes containing lithium heparin anticoagulant. The blood tubes were immediately placed in ice and then centrifuged within 1 h of collection and plasma transferred to cryovials and stored at -80°C until shipment to the University of Rhode Island. The animal study protocol was in compliance with Louisiana State University Institutional Animal Care and Use Committee (IACUC protocol # 16-017).

A 10μL of the internal standard, urolithin B (6) solution at 10,000 ng/mL was spiked into 100μL of equine plasma sample at each of the aforementioned time point and were vortexed for 10 s. The samples were vortexed for 30 s after which 200μL acetonitrile was added for protein precipitation. After allowing the samples to sit at 4°C for 10 min, the mixtures were centrifuged for 10 min. The supernatant was collected and diluted with 100μL DI water containing 0.5% (v/v) formic acid then subjected to LC-ESI–MS/MS analysis.

3. Results and discussion

3.1. Optimization of LC–MS/MS conditions

For this study, a Waters XBridge BEH C18 column was employed for liquid chromatography which, in combination with the selected solvent mobile phase, provided symmetrical peaks with high intensity and selectivity. The mass spectra of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and the internal standard, urolithin B (6) were acquired by direct infusion of their respective solutions on a QTRAP 4500 mass spectrometer (MS) coupled with an electrospray ionization (ESI) source under both positive and negative ion modes. The individual solutions were prepared by mixing 10 g/mL of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) or, the internal standard, urolithin B (6), in 43% aqueous acetonitrile containing 0.1% (v/v) formic acid. The MS operating in negative mode showed better sensitivity compared to the positive mode. The full scan mass spectra of compounds 1, 2, 3, 4, 5, and 6 showed [M-H]- ions of m/z 367.1, 337.1, 307.1, 447.1, 543.2, and 211.0, respectively Fig. 2A, The [M-H]- ions of compounds 1-6 were subjected to collision induced dissociation at an optimal collision energy. The most abundant daughter ions were 134, 119, 119, 134, 134, and 167 for curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and, the internal standard, urolithin B (6), respectively (Fig. 2B). The levels of the analytes were monitored by following ion transition pairs at m/z values of curcumin (1), 367/134, demethoxycurcumin (2) 337/119, bisdemethoxycurcumin (3) 307/119, curcumin-O-sulfate (4) 447/134, curcumin-O-glucuronide (5) 543/134 and the internal standard, urolithin B (6) 211/167. Details of the optimization of the mass spectrometry are shown in Table 1.

It is well known that the major fragmentation of curcumin-O-glucuronide (5) is glycosidic cleavage [13] leading to m/z 367 which corresponds to curcumin (1), Similarly, curcumin-O-sulfate (4) also undergoes cleavage to form m/z 367 corresponding to curcumin (1), Therefore, the LC method was optimized to obtain optimal peak resolution and separation of all five of the analytes in the shortest elution time. The best resolution of peaks was achieved with acetonitrile/water (43:57, v/v with 0.1% formic acid) at a flow rate of 0.2 mL/min. This resulted in retention times of 2.1, 3.1, and 7.0 min for curcumin-O-glucuronide (5), curcumin-O-sulfate (4), and curcumin (1), respectively. Therefore, to quantify curcumin (1), we used the MRM transition ion pair (367/134) of the curcumin (1), peak at retention time of 7.0 min. Using these aforementioned optimized LC conditions, the retention times for the other three analytes, demethoxycurcumin (2), bisdemethoxycurcumin (3) and the internal standard, urolithin B (6), were 6.7, 5.4, and 3.2 mins, respectively.

3.2. Method validation

3.2.1. Specificity

Representative chromatograms of blank equine plasma and blank equine plasma spiked with curcumin (1), demethoxycurcumin 2, bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) (each at 50 ng/mL), and the internal standard, urolithin B (6) (at 1000 ng/mL) are shown in Fig. 3. No interference peaks were observed in any of the five analytes and the internal standard, urolithin B (6) retention times due to endogenous matrix components.

The retention times of the five analytes, curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and, the internal standard, urolithin B (6), were 7.0, 6.7, 5.4, 3.1, 2.1, and 3.2 min, respectively. As aforementioned, curcumin (1) was quantified by using the ion transition pair of MRM 367/134 of the peak at 7.0 min thereby distinguishing it from the equivalent MRM 367/134 of curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) which eluted at retention times of 3.1 and 2.1 min, respectively. This indicated the efficient specificity of the method.

3.2.2. Calibration curves and lower limit of quantification (LLOQ)

All calibration curves were linear using weighted linear least square regression 1/X2. The calibration curves for curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3) and curcumin-O-sulfate (4) showed linearity over the concentration range of 0.5–1000 ng/mL in equine plasma. The calibration curves for curcumin-O-glucuronide (5) showed linearity over the concentration range of 1–1000 ng/mL in equine plasma. Based on the data, the developed method showed good linearity reliability.

3.2.3. Accuracy and precision

The intra-day and inter-day accuracy and reproducibility of the analytes in equine plasma were evaluated at concentrations of 1, 10, 100, and 500 ng/mL for curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3) and curcumin-O-sulfate (4) and at concentrations of 2, 10, 100, and 500 ng/mL for curcumin-O-glucuronide (5), The results are summarized in Table 2. All of the QC samples for the intra-day and inter-day validation showed CV errors of less than 15% and accuracy between 85% and 115%. These precision and accuracy values are acceptable according to the FDA criterion of a GLP analytic method validation [23]. This confirmed that the current method has a satisfactory accuracy, precision, and reproducibility for the simultaneous quantification of all of the five analytes over a broad concentration range.

3.2.4. Matrix effects and recovery

The mean extraction recoveries and matrix effects for all of the five analytes and the internal standard, urolithin B (6) in equine plasma at three different concentrations are shown in Table 3. No significant loss of analytes was due to sample processing. The matrix effect on the determination of the analytes was within the acceptance criteria at each level evaluated.

Fig. 2.
A The full scan mass spectra of the deprotonated molecular ions of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and the internal standard, urolithin B (6) under the negative mode. B. MS/MS spectra of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and the internal standard, urolithin B (6).
Fig. 3.
Representative MRM chromatograms of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and the internal standard, urolithin B (6) in equine plasma (I), blank equine plasma spiked with the five analytes at 50 ng/mL and the internal standard, urolithin B (6) at 1000 ng/mL(II).

3.2.5. Stability

The stabilities of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5), at concentrations of 10, 100 and 500 ng/mL, were evaluated in equine plasma. All stability conditions tested had an acceptance criterion of less than 15% relative error (RE) difference. All of the analytes were stable in 0, 3, 6, and 9 h post-extraction in the auto-sampler in storage at -80°C for 15 days, in three freeze-thaw cycles, and in stock solutions stored at -80°C for 15 days. The stability data are summarized in Table 4.

3.3 Quantification of curcumin-O-sulfate (4) plasma levels in equines

The method described above was applied to measure plasma levels of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) in plasma samples collected from two horses after oral dosing (for two weeks) of a supplement containing curcuminoids (Longvida®).

Table 2
The vaildation parameters: intra-day and inter-day accuracies and precisions of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) in equine plasma.
Table 3
The validation parameters: recovery and matrix effect of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4), curcumin-O-glucuronide (5) and the internal standard, urolitin B (6) in equine plasma.
Table 4
The validation paramters: stability of curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) in equine plasma.

Plasma samples collected on day 1 from both horses before and after supplementation did not show the presence of any of the curcuminoids nor their metabolites. However, after 14th daily oral dose, plasma from both horses showed quantifiable levels of curcumin-O-sulfate (4), As shown in Fig. 4, the curcumin-O-sulfate (4) plasma levels of equine I reached a Cmax of 2.4 ng/mL at 60 min and was detectable up to 2.5 h. In equine II, curcumin-O-sulfate (4) had a Cmax of 1.2 ng/mL at 105 min and was detectable up to 5 h after the 14th oral dose.

Fig. 4.
The plasma concentration-time profiles of curcumin-O-sulfate (4) after an oral dose (for two weeks) of a supplement containing curcuminoids (Longvida®) in two horses.

As aforementioned, the metabolism of curcuminoids in biological samples from rodents [9–12] and humans [13–15,19] have been previously studied. While curcumin-O-sulfate and curcumin-O-glucuronide have been detected in the plasma of humans and rodents, in the current study only curcumin-O-sulfate was detected in the plasma of horses. However, further studies using a larger number of subjects (n>2 horses) and different doses of curcumin are warranted to increase scientific knowledge on the metabolism of this natural product by horses.

4. Conclusions

In conclusion, a sensitive, optimized, and validated LC-ESI–MS/MS method was developed for the simultaneous determination and quantification of five analytes, curcumin (1), demethoxycurcumin (2), bisdemethoxycurcumin (3), curcumin-O-sulfate (4) and curcumin-O-glucuronide (5) in equine plasma. This method provides a comprehensive analytic method to characterize the pharmacokinetics of curcumin-O-sulfate (4) in equine plasma after the oral ingestion of curcumin. This is the first report for the simultaneous determination of these five analytes in equine biological samples. Our group’s future studies will include the utilization of the validated LC-ESI–MS/MS method developed herein for the detection and quantification of the aforementioned analytes in biological samples collected from horses treated with the curcumin formulation (Longvida®). We are also planning on conducting further studies with the curcumin formulation to evaluate the presence and levels of free curcuminoids and their metabolites in buffy coat and red blood cells of horses and human subjects which has not been previously studied.

Acknowledgements

This project was conducted using a mass spectrometry instrument located in the Rhode Island NSF EPSCoR research facility, supported in part by the National Science Foundation EPSCoR Cooperative Agreement#EPS-1004057.

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