Simultaneous Determination of Cis- and Trans-palmitoleic Acid in Rat Serum by UPLC-MS/MS

doi:10.21203/rs.3.rs-1619071/v2

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Simultaneous Determination of Cis- and Trans-palmitoleic Acid in Rat Serum by UPLC-MS/MS

https://doi.org/10.21203/rs.3.rs-1619071/v2

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Background: Cis-palmitoleic acid (cPOA) and trans-palmitoleic acid (tPOA) are isomers of palmitoleic acid, which are monounsaturated fatty acids, affecting glucose and lipid metabolism, and reducing insulin resistance. tPOA was better than cPOA in regulating lipid metabolism in hyperlipidemia mice, but the pharmacokinetic, metabolic transformation and structure-activity relationship have not been reported.

Method: A precise and accurate ultra performance liquid chromatography-tandem mass spectroscopy (UPLC–MS/MS) method was developed to determine cPOA and tPOA simultaneously. cPOA and tPOA were administered i.g. (intragastric gavage) to rats at 75 mg/kg, serum samples were analyzed by UPLC-MS/MS on a reverse-phase BDS C18 column equilibrated and eluted with water (A) and acetonitrile (B) at a flow rate of 0.3 mL/min.

Results: The calibration curves for cPOA and tPOA were linear over the range 0.1~12 μg/mL. Analytes were monitored by selected-reaction monitoring in negative electrospray ionization mode. The Tmax of cPOA was 0.94±0.44 h and the Cmax 8.17±1.97 μg/L, and the Tmax of tPOA was 1.50±0.98 h and the Cmax 14.77±11.91 μg/L. AUC_0-24h of cPOA and tPOA were 59.45±29.83 and 113.88±72.25 mg/L*h.

Conclusions: The method was applied in pharmacokinetic study of cPOA and tPOA in rat serum successfully. Besides, it’s found the concentration of cPOA and tPOA fluctuated in serum of rats with the consistent trend, which may be reciprocal bio-convert in the body.

Cis-palmitoleic Acid (cPOA)

Trans-palmitoleic Acid (tPOA)

Metabolite

UPLC-MS/MS

Pharmacokinetics

Palmitoleic acid (POA, C16:1, n-7) is an omega-7 monounsaturated fatty acid in plant and fish oil [1]. Cis-POA (cPOA) and trans-POA (tPOA) are isomers with different space structures(Fig. 1). cPOA is common in natural POA, which has been widely reported. It has been demonstrated that cPOA may influent favorably glucose and lipid metabolism through multiple mechanisms [2–3]. cPOA affected the key enzymes of blood glucose metabolism, regulated insulin secretion and reduced insulin resistance [4–5]. Orally administered cPOA induced satiety, enhanced the release of satiety hormones and decreased food intake in mice [6], in addition, cPOA reduced body weight, ameliorated the development of hypertriglyceridemia and hyperglycemia, and improved insulin sensitivity [7]. However, trans-fatty acid is the subject of an ongoing discussion on both suggested positive and negative associations with cardiovascular and metabolic risk factors [8]. While higher tPOA proportion in plasma phospholipids improved insulin sensitivity or decreased onset of type 2 diabetes disease [9]. tPOA regulated glycolipid metabolism, closely related to low density lipoprotein [10–12], which has been used as a biomarker for reducing the risk of coronary heart disease and type 2 diabetes. Most of the reports which analysed fatty acids or fatty acid profiles used GC-FID or GC-Ms, while, the detection pretreatment of the biological matrix requires separated and methylation of fatty acids before measured. Additionally, biological samples contain kinds of fatty acids, and the pretreat method may destroy the original molecular structure of fatty acids in the biological matrix and the result incomprehensive, besides, requring more biological sample size or content [13–14]. cPOA was determined in the subcutaneous fat of human body, and tPOA was determined in the 200 µL plasma of human by GC thorough, aminopropyl SPE columns for the separation (Isolute, Biotage) and transmethylated with methanolic-HCL into fatty acid methyl esters (FAME) [15–17], but there was no method reported to determine cPOA and tPOA in simultaneously. So, it’s important to develop a method for the qrauantitative analyses of cPOA and tPOA in biological samples for the studies of biological activity, drug delivery systems or regenerative medicine. In the present paper, a UPLC-MS/MS method for quantification of simultaneous determination direct of cPOA and tPOA in rat serum was developed. This method proved to be selective and sensitive, with a wide range of detectionand low limit of detection, used to ascertain the phar-macokinetics of cPOA and tPOA.

2.1 Materials and animals

cPOA and tPOA were purchased from NU-CHEK (༞99%, Elysian, USA). HPLC-grade formic acid was obtained from Roe Scientific Inc. (Powell, OH, USA). HPLC-grade acetonitrile, methanol and MTBE(methyl tert-butyl ether)were obtained from Merck KGaA (Darm-stadt, Germany). Ultrapure water was from a Millipore Milli-Q system (Millipore Corp., Billerica, MA, USA). The other solvents or reagents were commercially available and reagent grade.

The use of rat approved by the Experimental Animal Ethics Committee of Nanjing University of Traditional Chinese Medicine in accordance with NIH guidelines. Ethical approval number: 201811A027. The authors complied with the ARRIVE guidelines. The 12 male Sprague-Dawley rats weighing 280 ± 20 g were maintained with 12 h:12 h light/dark cycles in a temperature-controlled room throughout the study. Standard rat chow and tap water were supplied ad libitum. Rat serum was collected from vein of rat after narcotized by intraperitoneal injection of pentobarbital sodium (Produlab Pharma B.V., 40 mg/kg) .

2.2 Chromatographic and mass spectrometric conditions

2.2.1 Liquid chromatography conditions

Analyte separations were performed on an Agilent UPLC-1290 system (Agilent Corp., Milford, MA, USA) using a BDS C18 column (2.1×100 mm, 2.1 µm, Thermo Fisher Scientific, USA). The mobile phase was included water (A) and acetonitrile (B) (A:B = 20:80, v/v) at a flow rate of 0.3 mL/min, and the injection volume was 1 µL.

2.2.2 Mass spectrometric conditions

Identification of cPOA and tPOA in serum samples was conducted using an AB 5500 Q-trap UPLC-MS/MS (ABSCIEX, Framingham, MA, USA) equipped with electro spray ionization (ESI). Quantitative analysis of cPOA and tPOA were also performed by UPLC-MS/MS. Detection was performed in negative ion mode under the following conditions: curtain gas at 35.0 L/h, and ion source gases at 50 L/h. AB Analyst 1.6.0 software (ABSCIEX, Framingham, MA, USA) was used for system control and data acquisition. ESI-MS/MS parameters are shown in Table 1. Detection was performed in the negative ion mode and conditions for cPOA and tPOA detection optimized using standards. Product ions obtained from deprotonated molecular ions of cPOA and tPOA included three main ions from each compound at m/z 235.2, 126.9, 111.0 and at m/z 234.7, 127, and 111.1, respectively.

Table 1

Electrospray ionization ESI-MS/MS parameters for cPOA and tPOA.
Analyte	Precursor Ion (m/z)	Daughter Ion (m/z)	Dwell Time (s)	DP(V)			CXP(V)
cPOA	253.2	235.2	20	-100	-8.2	-25.8	-11.0
	253.2	126.9	20	-100	-8.2	-31.0	-11.0
	253.2	111.0	20	-100	-8.2	-29.0	-11.0
tPOA	253.2	234.7	20	-100	-7.3	-28.0	-8.5
	253.2	127.0	20	-100	-7.3	-26.0	-12.0
	253.2	111.1	20	-100	-7.3	-26.0	-12.0

2.3 Stock solutions and working solutions

Individual standard stock solutions of cPOA and tPOA (2.50 mg/mL, 2.24 mg/mL respectively) were prepared in acetonitrile. These stock solutions were serially diluted with acetonitrile to provide standard working solutions in the concentration range of 0.175 ~ 42.0 µg/mL for cPOA and tPOA. All solutions were stored at -20℃ and brought to room temperature before use.

2.4 Calibration standard curves and QC samples

Calibration standard (CS) curves were prepared by spiking 20 µL of the appropriate analyst working solution into 50 µL of blank rat serum. The effective concentrations were 0.1, 0.5, 2.5, 5, 10, 12 µg/mL for cPOA and tPOA. QC samples were prepared as compound samples for each concentration at 0.5 µg/mL for cPOA and tPOA, and stored at -20℃ until use. Rat serum samples, serving as QCs, were processed the following sample procedure as for unknown samples.

2.5 Serum sample preparation

50 µL of serum sample (blank, or pharmacokinetics serum sample) in a 2.0 mL centrifuge tube, 60 µL of aqueous solution with formic acid in 5%, 100 µL methanol, and 1250 µL MTBE were added and mixed by vortexing for 3 min. After centrifugation at 18,000 ×g for 10 min, the clear supernatant of 1 mL was extracted to a new centrifuge tube, blowed by flowing nitrogen, and redissolved by 200 µL acetonitrile solution. Centrifugation at 18,000 ×g for 10 min again, supernatant was injected into the UPLC-MS/MS system.

2.6 Method validation

Assay validation performed was based on the currently accepted FDA prescription and per guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use [18]. Each blank serum sample was processed through the extraction procedure and tested to ensure no rat serum interference with the analyte. While the serum sample preparation was 60 µL of aqueous solution with formic acid in 5%, 100 µL methanol, and 1250 µL MTBE were added and mixed by vortexing for 3 min. After centrifugation at 18,000 ×g for 10 min, the clear supernatant of 1 mL was extracted to a new centrifuge tube, blowed by flowing nitrogen, and redissolved by 200 µL acetonitrile solution.

The determination of the extraction recoveries of cPOA and tPOA was at three QC concentrations. And the calculation of the recoveries was by comparing analyte peak area ratios for each analyte in serum samples with those of analytes in the serum matrices by extracting analyte-free serum samples which were prior to chromatography. In extracted rat serum, matrix effects from endogenous substances were presented, which might have caused ion signal suppression or enhancement. Matrix effects at three QC concentrations (0.5, 2.5, and 10 ng/mL) were measured by comparing peak responses of samples post-extraction (A) with that of pure standard solution which contained equivalent amounts of the two compounds (B). The ratio (A/B × 100%) was used to evaluate the matrix effect and the extraction recovery and matrix effect of cPOA and tPOA were evaluated simultaneously by the same method.

During sample storage and processing procedures, the stability of cPOA and tPOA in rat serum was assessed by analyzing replicates (n = 6) of three QC concentrations. The freeze–thaw stability was determined through three freeze–thaw cycles. All stability testing of QC samples were determined according to calibration curves of freshly prepared standards.

2.7 Pharmacokinetic studies of cPOA and tPOA

Male rats (ICR, 280 ± 20 g) were obtained from the Laboratory Animal Center of Nanjing University of Chinese Medicine (Nanjing, China). Animal handling procedures followed standard operating procedure approved by the institutional animal care and use committee. All rats were dosed following overnight fasting except for water ad libitum. For pharmacokinetic studies, 18 male rats were randomly divided into three groups. In the first group, rats were administered intragastric gavage of normal saline with 75 mg/kg body weight. Blood samples were collected at the time points of 0, 10, 20, 30, 40, 60 min, and 2 h, 3 h, 6 h, 12 h, 24 h. Rats in the second group were administered i.g. of cPOA with 75 mg/kg body weight. Serial blood samples were collected in tubes via the orbital venous plexus before and at time points of 0, 10, 20, 30, 40, 60 min, 2 h, 3 h, 6 h, 12 h, 24 h, after administration. In the third group, rats were administered intragastric gavage of tPOA with 75 mg/kg body weight. Blood samples were collected at the time points of 0, 10, 20, 30, 40, 60 min, and 2 h, 3 h, 6 h, 12 h, 24 h. Serum was separated and stored frozen at -80℃ until analysis. The following main pharmacokinetic parameters were analyzed using the non-compartmental pharmacokinetics data analysis soft-ware of PK solution 2TM (Summit Research Service, Montrose, CO, USA): area under curve from zero to the last measurable serum concentration point (AUC_0 − t, t = 24h), maximum concentration (Cmax), time-to-maximum concen-tration (Tmax).

3.1 Determination of cPOA and tPOA by UPLC-MS/MS

cPOA and tPOA were determined by ultra performance liquid chromatography ESI tandem mass pectrometry (UPLC-MS/MS). The total chromatography time was 8.0 min and retention time cPOA and tPOA were 3.86 min and 4.23 min, respectively (Fig. 2A, B, C). cPOA and tPOA were detected in serum samples intragastric administration. Both cPOA and tPOA were detected simultaneously in samples after pretreatment. The results showed that the blank serum might contain trace amounts of cPOA and tPOA (Fig. 2D, E).

3.2 Method Validation

3.2.1 Selectivity and Specificity

Selectivity and specificity were investigated by comparing chromatograms of 6 different blank rat serum samples to corresponding spiked serum samples. No other endogenous substances were observed to interfere with cPOA and tPOA in any samples, but there was certain amount of cPOA and tPOA in blank serum. Specificity was verified by comparing retention times of cPOA and tPOA (3.86 and 4.23 min, respectively) in quality control (QC) samples (n = 6), which indicated differences of less than 5%.

3.2.2 Calibration Curve Linearity, Limit of Detection(LOD), and Lower Limit of Quantification༈LLOQ༉

Standard curves were obtained by plotting the ratios of peak areas of cPOA or tPOA. The curves showed correlation coefficients greater than 0.999 and exhibited good linearity over concentration ranges of 0.1 ~ 12 µg/mL for cPOA and tPOA. Typical calibration equations were y = 2.39e^+ 005x (R = 0.9991) for cPOA, and y = 9.06e^+ 004x (R = 0.9999) for tPOA, where y represented the peak area ratio of an analyte, and x represented an analyte concentration. The limit of detection (LOD) was estimated to be 30 ng/mL. The lower limits of quantification (LLOQ) for both cPOA and tPOA were defined as 0.1 µg/mL.

3.2.3 Accuracy and precision

The method’s accuracy and precision were summarized in Table 2. Accuracy was required to be within ± 15% (20% for LLOQ) and precision not to exceed ± 15% (20% for LLOQ), following criteria for biological sample analysis according to U.S. Food and Drug Administration (FDA) guidelines[19]. The present results suggested that the method was accurate and precise for simultaneous analysis of cPOA and tPOA in rat serum samples.

Table 2

Accuracy and precision for determination of cPOA and tPOA in serum samples (n = 6).
Analyte	Concentration (µg/mL)	Mean ± SD (µg/mL)	Accuracy (%)	Precision (%)
cPOA	0.50	0.56 ± 0.15	111.0	0.98
	2.50	2.76 ± 0.86	110.4	1.45
	10.0	8.54 ± 1.79	85.4	2.01
tPOA	0.50	0.54 ± 0.16	109.0	1.48
	2.50	2.64 ± 0.65	105.6	2.13
	10.0	8.15 ± 0.98	81.5	4.07

3.2.4 Recovery and Matrix Effects

The recoveries of cPOA and tPOA spiked into rat serum were determined at three QC concentrations. The recoveries of cPOA were 101.43 ± 1.37, 102.11 ± 1.25, and 101.92 ± 1.84 (n = 6) at concentrations of 0.5, 2.5 and 10 µg/mL, and those of tPOA were 98.28 ± 1.23, 100.66 ± 1.82, and 99.75 ± 3.01 (n = 6), respectively (Table 3). The peak area of the standard analyte spiked into blank serum, minus that in the blank serum, were compared with that of injecting the standard analyte of 0.5, 2.5 and 10 µg/mL for cPOA and tPOA. In terms of matrix effects, all the ratios defined above were within acceptable limits (89.51% – 94.72%) (Table 3). No significant matrix effect for cPOA and tPOA were observed, showed that ion suppression or enhancement from serum components was negligible for this method.

Table 3

Recovery and matrix effects of cPOA and tPOA in serum samples (n = 6).
Analyte Concentration (µg/mL)		Matrix Effects		Recovery
		Mean ± SD (µg/mL)	RSD (%)	Mean ± SD (µg/mL)	RSD (%)
cPOA	0.50	94.12 ± 1.91	2.96	101.43 ± 1.37	1.39
	2.50	92.64 ± 1.67	1.98	102.11 ± 1.25	1.31
	10.0	91.45 ± 2.43	2.56	101.92 ± 1.84	2.09
tPOA	0.50	94.72 ± 1.05	1.27	98.28 ± 1.23	2.11
	2.50	93.93 ± 2.47	2.69	100.66 ± 1.82	1.99
	10.0	89.51 ± 3.05	3.55	99.75 ± 3.01	3.58
RSD, relative standard deviation.

3.2.5 Stability

Results of stability tests indicated that the analytes were stable in the laboratory conditions (Table 4). The stability of cPOA and tPOA was evaluated as described in the experimental section.

Table 4

Stability of cPOA and tPOA (n = 6).
Storage Condition (-80℃)	cPOA			tPOA
Storage Condition (-80℃)	Concentration (µg/mL)	Accuracy (%)	RSD (%)	Concentration (µg/mL)	Accuracy (%)	RSD (%)
1 freeze-thaw cycle	0.50	111.0	1.27	0.50	109.0	1.27
	2.50	110.4	1.53	2.50	105.6	1.53
	10.0	85.4	2.42	10.0	81.5	2.42
2 freeze-thaw cycle	0.50	105.0	3.02	0.50	99.2	3.02
	2.50	107.6	2.98	2.50	101.4	2.98
	10.0	86.6	2.54	10.0	83.7	2.54
3 freeze-thaw cycle	0.50	96.4	2.84	0.50	103.2	2.12
	2.50	99.2	3.07	2.50	91.7	2.97
	10.0	82.7	3.87	10.0	85.9	3.26

3.3 Pharmacokinetic analyses of cPOA and tPOA

The mean concentrations of cPOA in blank rat serum fluctuated around 5 µg/mL (4 ~ 8 µg/mL) in 24 hours, while, the mean concentrations of tPOA fluctuated around 2 µg/mL (0 ~ 4 µg/mL) in 2 hours, and lower concentration after 2 hours (Fig. 3), it may relate to the concentration of original cPOA in organism. The result showed that cPOA and tPOA concentrations fluctuated in the same trend in certain time.

The pharmacokinetic profiles of cPOA and tPOA were investigated by the method at 75 mg/kg i.g. dose of cPOA to rats (Fig. 4A). The mean concentration–time profiles of cPOA and tPOA in serum showed that cPOA was absorbed rapidly by rats such that it increased in serum in 0.3 h after administration and decreased slowly thereafter (Fig. 4A). The Tmax of cPOA was 0.94 ± 0.44 h and the Cmax 8.17 ± 1.97 µg/L (Table 5). The concentration of cPOA was decreased to the level of blank serum rapidly. Meanwhile, tPOA was detected simultaneously after cPOA dosing, mean serum concentration–time profiles of tPOA fluctuated (4 ~ 7 µg/L) in the same trend with cPOA. And, the concentration of tPOA in serum was retain 4 µg/L to 6 µg/L.

Table 5

Pharmacokinetic parameters of baseline-corrected cPOA and tPOA in rats after i.g. administration of cPOA and tPOA at 75 mg/kg body weight.
Parameters	Unit	Mean ± SD
Parameters	Unit	cPOA	tPOA
C_max	mg/L	8.17 ± 1.97	14.77 ± 11.91
T_max	h	0.94 ± 0.44	1.50 ± 0.98
AUC(0-24h)	mg/L*h	59.45 ± 29.83	113.88 ± 72.25

The pharmacokinetic profiles of cPOA and tPOA were investigated following a single i.g. dose at 75 mg/kg body weight of tPOA to rats (Fig. 4B). The mean serum concentration–time profiles of cPOA and tPOA showed that tPOA was absorbed slowly by rats such that it increased in serum in 2 h after administration and decreased rapidly thereafter (Fig. 4B). The Tmax of tPOA was 1.50 ± 0.98 h and the Cmax 14.77 ± 11.91 µg/L. tPOA was decreased to the concentration of original level rapidly. Meanwhile, cPOA was detected after tPOA dosing simultaneously, mean serum concentration–time profiles of cPOA fluctuated (5 ~ 15 µg/L) in the same trend with cPOA. And, the concentration of cPOA in serum was maintain 10 µg/L, which was more than original concentration of cPOA.

The pharmacokinetic analysis has been made on cPOA and tPOA, both with pre-existing endogenous levels (Fig. 3). Therefore, the data should be evaluated corrected for pre-existing baseline levels. The endogenous concentrations of cPOA and tPOA were variable, therefore, it contributed to the total concentration of cPOA and tPOA, and added to the inter- and intra-individual variability. Therefore, a pre-dose adjustment of measured levels was conducted. For each rat and test period, the pre-dose concentrations were subtracted from the measured serum concentrations, and the pharmacokinetic parameters was calculated on the baseline-adjusted concentrations according to regulatory guidelines for bioequivalence testing of endogenous substances [20, 21]. If the measured value was lower than the pre-dose concentration the adjusted concentration was set to zero (Fig. 5).

The pharmacokinetic parameters were determined using non-compartmental analysis from the serum concentrations of cPOA and tPOA. The area under the serum concentration-time curve (AUC) was calculated by applying the log-linear trapezoidal model to the measured cPOA and tPOA concentrations and the combined sum of cPOA and tPOA at the actual sampling time points. Because of the fluctuated pre-existing endogenous levels, it was not possible to define a terminal elimination phase. The elimination rate constant was therefore not calculated, and exposure was reported as the AUC from time 0 to 24 h post-dosing (AUC_{0 − 24h}). AUC_{0 − 24h} of cPOA and tPOA were 59.45 ± 29.83 and 113.88 ± 72.25 mg/L*h. The pharmacokinetic parameters of cPOA and tPOA were calculated and summarized in Table 4.

cPOA in organisms, especially in adipose tissue, may be related to the development of cardiovascular disease, tPOA has been used as a biomarker for reducing the risk of type 2 diabetes and coronary heart disease [10–12], which may be due to the correlation of cPOA to tPOA, playing a decisive role in biological activity in the body.

tPOA in the body was mainly obtained from food intake and endogenous oxidation reaction. Isooleic acid (VA, 18:1, t11) in food could be converted into tPOA (16:1, t9) by shortening the chain length, with a conversion rate of 17% [9]. As the concentration of tPOA in daily diet was relatively low and easy to be oxidized, tPOA in serum was mainly derived from endogenous transformation of VA from dietary [22]. By simulating the differentiation of adipocytes in vitro, it was found that the increasing content of cPOA would increase the content of VA and conjugated linoleic acid (CLA, 18:2, c9, t11) in adipocytes [23]. Therefore, it could be inferred that exogenous cPOA (16:1, c9) may generate VA (18:1, t11) by lengthening carbon chain and desaturation during metabolism in vivo, and further convert to tPOA (16:1, t9) by shortening chain length (Fig. 6). Huang [24–25] selected healthy adults to study the effect of oral seabuckthorn fruit oil (main ingredient cPOA, without tPOA) on the content changes of tPOA and tPOA phosphatide fatty acids in fasting serum, and found that the content of tPOA in fasting serum increased with high oral dose of cPOA (1520 mg/d) for 3 consecutive weeks (P = 0.0668). In our pharmacokinetic study, it was found that the change of exogenous cPOA concentration in serum leaded to the fluctuation of (endogenous) tPOA concentration with the consistent trend, indicated the possibility of cPOA being metabolized into tPOA in vivo. Also, we found the change of (endogenous) cPOA concentration in serum was correspondent with the fluctuation of exogenous tPOA concentration. So, it’s suggested that cPOA and tPOA in serum may interconvert of isomeric compound configurations. The pretreatment process of GC method was cumbersome and has a long total chromatography time and retention time. Establishing rapid, accurate and simultaneous detection of cPOA and tPOA in biological samples has important research value for laboratory, clinical research and real-time detection cPOA and tPOA of organism. The method may be applied to clinical investigations to elucidate the biological activity and structure-activity relationship (SAP) of cPOA and tPOA in humans. And favoring the development of biological activity, drug delivery systems or regenerative medicine to define the metabolism and transformation between fatty acids. Besides, previous studies have shown that tPOA was better than cPOA in regulating lipid metabolism in hyperlipidemia mice [26]. It was suggested that cPOA and tPOA in serum may interconvert of isomeric compound configurations. Therefore, whether cPOA played a role in regulating lipid metabolism by transforming into tPOA, and the bioconversion relationship between cPOA and tPOA and the conversion rate could be further studied by isotope labeling or fluorescence labeling method.

A sensitive and rapid UPLC-MS/MS method for determination of cPOA and tPOA simultaneously in rat serum was established, which were identified in serum after i.g. administration. And the method was used to a pharmacokinetic study of cPOA and tPOA in rats. The result suggested that the endogenous cPOA concentration in serum was correspondent with the fluctuation of exogenous tPOA concentration, and cPOA and tPOA in organism may interconvert of isomeric compound configurations, which of the metabolic transformation mechanism needs further investigation.

POA: Palmitoleic acid, cPOA: Cis-palmitoleic acid, tPOA: Trans-palmitoleic acid, UPLC-MS/MS: Ultra Performance liquid chromatography-tandem mass pectrometry.

Consent for publication

Not applicable.

Availability of data and materials

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

All authors declare no conflict of interest.

Funding

This work was supported by the National Natural Science Fund, China [grant number 81870138], PhD Scientific Research Fund of Binzhou University [grant number 2021Y15], Natural Science Foundation of Shandong Province [ZR2019MH054].

Author contributions

Wenwen Huang, Yiping Zhang and Liping Zhong carried out the experiment and prepared the manuscript. Yiping Zhang, Liping Zhong, Chunlong Sun contributed with valuable discussions and scientific input. Wenwen Huang conducted the experimental design. All authors participated in preparing the manuscript and approved the final version.

Conflicts of Interest

All authors declare no conflict of interest.

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No competing interests reported.

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Simultaneous Determination of Cis- and Trans-palmitoleic Acid in Rat Serum by UPLC-MS/MS

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1. Introduction

2. Materials And Methods