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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 3  |  Issue : 4  |  Page : 191-196

Determination of periplocymarin in human blood and urine by high-performance liquid chromatography-mass spectrometry


Department of Forensic Toxicological Analysis, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, China

Date of Web Publication11-Jan-2018

Correspondence Address:
Prof. Liao Lin Chuan
West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jfsm.jfsm_46_17

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  Abstract 

A simple, rapid and sensitive liquid chromatography with tandem mass spectrometry method for the determination of periplocymarin in human blood and urine was developed. The digoxin-d3 was used as an internal standard. Periplocymarin and digoxin-d3 (IS) were processed with ethyl acetate by liquid-liquid extraction. The chromatographic separation was performed on a Shim-pack XR-ODSIII C18 column with a 7 min gradient elution using methanol-ammonium formate (5 mmol/L) as mobile phase at a flow rate of 0.3 mL/min (65:35, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer using positive-ion mode electrospray ionization in selected reaction monitoring mode. The periplocymarin was well separated from the internal standard. Two calibration curves were linear within the concentration range 0.01-1 μg/mL. The limit of detection and quantification of blood and urine samples were both estimated at 0.005 and 0.01 μg/mL. The interday and intraday precisions, accuracy, and recovery were assessed to verify this method. The results showed that the method was suitable for the determination of periplocymarin in forensic toxicological analysis and clinical diagnosis.

Keywords: Blood, forensic toxicological analysis, liquid chromatography-tandem mass spectrometry, periplocymarin, urine


How to cite this article:
Xia CJ, Tao WQ, Fan C, Hao W, Qing FY, Gui WZ, Hua DX, Yi Y, Yi YY, Chuan LL. Determination of periplocymarin in human blood and urine by high-performance liquid chromatography-mass spectrometry. J Forensic Sci Med 2017;3:191-6

How to cite this URL:
Xia CJ, Tao WQ, Fan C, Hao W, Qing FY, Gui WZ, Hua DX, Yi Y, Yi YY, Chuan LL. Determination of periplocymarin in human blood and urine by high-performance liquid chromatography-mass spectrometry. J Forensic Sci Med [serial online] 2017 [cited 2018 Jul 18];3:191-6. Available from: http://www.jfsmonline.com/text.asp?2017/3/4/191/222891


  Introduction Top


Periplocin, the major active component isolated form Cortex periplocae, has anticancer,[1] antirheumatism,[2] and other pharmacological effects. The mechanism of cardiovascular effect and strengthening cardiotonic function for periplocin has been reported.[3],[4] Periplocymarin was a major pharmacologically active product by periplocin.[5] It has been reported that periplocymarin showed significant cardiotonic effect which was faster and stronger than the prototype drug periplocin.[6] After entering the body, periplocin would be quickly metabolized into periplocymarin.[7] Hence, it could be detected from human blood and urine. However, as a strong cardiac drug, the periplocymarin has been used in the treatment of heart failure. The poisoning mechanism of periplocin and periplocymarin was similar with digoxin and other cardiac glycosides.[8] Due to narrow treatment concentration range, it is easy to overdose and inhibit myocardial enzyme activity, resulting in various arrhythmia and death.[9] With the increasing of heart failure disease, the death cases caused by Chinese herbal medicine and cardiac glycoside poisoning increased year by year. Recently, several analytical methods for periplocin and periplocymarin have been reported, such as high-performance liquid chromatography (HPLC)[9] and HPLC-mass spectrometry (MS).[10],[11],[12] However, HPLC was mainly used for vitro samples, and LC-MS was used to explore the pharmacokinetics of periplocymarin in rat plasma. Few of analytical methods were used for human blood and urine. Based on LC-MS, a method to qualify and quantify periplocymarin in blood and urine was established. It was recommended to be applied to treatments of clinical periplocin or periplocymarin poisoning and forensic toxicological analysis.


  Subjects and Methods Top


Apparatus and reagents

A LC/MS-8030 (Shimadzu, Japan) chromatography fitted with an autoinjection system was applied. The data system contains the software required to collect chromatography and spectra. Chromatographic separation was achieved on a Shim-pack XR-ODSIII C18 column (Shimadzu, Japan) (2.0 mm × 75 mm, 1.6 μm).

A periplocymarin standard (purity ≥99%, 20 mg) was purchased from Desert Biological Technology Co., Ltd (Chengdu, China). The internal standard digoxin-d3 (IS, purity ≥93.4%, 1 mg) was purchased from Kangtai Biological Technology Co., Ltd (Guangzhou, China). Methanol (HPLC grade) was purchased from the Fisher Scientific company (US); Ethyl acetate (HPLC grade) was purchased from Kelong Chemical Reagent Factory (Chengdu, China); Ammonium formate (analytical grade, purity 97%) was purchased from (ANPEL, USA). The chemical structures of the analytes were shown in [Figure 1].
Figure 1: The chemical structures of the digoxin-d3 (a), periplocin (b), and periplocymarin (c)

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Instrumental conditions

High-performance liquid chromatography conditions

A LC-MS analysis was performed using the autoinjection system with a Shim-pack XR-ODSIII C18 column (Shimadzu, Japan) (2.0 mm × 75 mm, 1.6 μm). Methanol-5 mmol/L ammonium formate (65:35, v/v) was used to achieve a desired separation at a flow rate of 0.3 mL/min. The gradient program was as follows: 0-3 min, 65% B, 3.01 min, 65%-50% B, 3.02-5 min, 50% B, 5.01 min, 50%-15% B,5.02-7 min, 65% B.

Mass spectrometry conditions

The mass spectrometer was operated in the positive electrospray ionization mode. The optimized MS parameters were designed as follows: capillary voltage at 4500V, nebulizing gas flow rate at 3 L/min; drying gas flow rate at 15 L/min, heat block temperature at 400°C, temperature at 160°C, and oven temperature at 30°C. Compound-dependent parameters are listed in [Table 1].
Table 1: Mass spectra properties of the compound and IS

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Working solution preparation

Standard stock solutions of periplocymarin and digoxin-d3 (IS) were weighed and dissolved in methanol at a concentration of 0.5 mg/mL; working solutions at concentration of 0.05, 0.125, 0.25, 0.5, 1.25, 2.5, 5 μg/mL and IS stock solution at 0.5 μg/mL were prepared through sequential dilution of the standard solutions with methanol. All solutions were stored at 4°C until analyzed.

Sample preparation

Samples were processed according to the following liquid-liquid extraction procedure: a 0.5 mL aliquot of each sample was transferred to a 10 mL glass tube and 100 μL of IS solution was added into the 10 mL centrifuge tube and then the mixture was vortex for 30. After the addition of 4 mL ethyl acetate, the sample was placed on a vortex mixer shaking for 5 min. After centrifugation at 3500 rpm for 5 min, the supernatant layer was transferred to a clean glass tube and evaporated to dryness under vacuum with nitrogen. The obtained residue was reconstituted with 500 μL of mobile phase, vortex mixed for 1 min, and centrifuged at 14,000 rpm for 5 min. Finally, 10 μL of the supernatant liquid was injected into the LC-MS system for analysis.


  Results and Discussion Top


Selection of the internal standard

The internal standard should be similar to the analyte in chemical and physical properties.[12] The structure of digoxin-d3 was similar to that of periplocymarin and it was not used in clinic, which effectively reduced the bias caused by the pretreatment of the analytical method. Hence, we selected digoxin-d3 as the internal standard.[10],[13]

Selection of the extraction method

Compared to literature,[14] our experimental subjects and objectives were different; we aim to establish an accurate analytical method for detecting periplocymarin from biological materials. However, in order to obtain better extraction efficiency, different types of extraction and dispersive solvents were analyzed in this study. Each extraction method was repeated three times within 1 day.

For the selection of the optimal extraction solvent, trichloromethane, ethyl acetate, acetone, and acetonitrile were investigated to extract periplocymarin from human blood and urine. By comparing the four extraction solvents, we found that ethyl acetate and trichloromethane had showed higher extraction efficiency than acetone and acetonitrile. However, considering the toxicity of trichloromethane can be harmful to long-term workers' health, the ethyl acetate was selected as the extraction solvent. We also tried different number of samples, different quantities of extraction solvents, and different flow rates of mobile phase; the results showed that the extraction recovery of method of our experiment which reached 91.2% was higher than that of the relevant literature.[14] Moreover, the higher the recovery rate, the more reliable the analysis results in the toxicological analysis. Therefore, 0.5 mL of samples, 4.0 mL of ethyl acetate, and a desired flow rate of 0.3 mL/min were selected as our research condition.

Selection of the optimum mobile phase

First, in order to get the most suitable mobile phase composition, by reviewing relevant literature,[11],[12],[13],[14],[15],[16] ultra-pure water including 0.1% (v/v) formic acid, ammonium acetate, ammonium acetate containing 0.1% (v/v) formic acid, ammonium formate, ammonium formate containing 0.1% (v/v) formic acid was used as the mobile phase A, respectively; acetonitrile, acetonitrile containing 0.1% (v/v) formic acid, methanol, methanol containing 0.1% (v/v) formic acid was used as mobile phase B, respectively. Meanwhile, we observed the ionization efficiency, peak type, and baseline stability of the analyte. By comparing different results, we found that when the mobile phase composition (ammonium formate [A] and methanol [B]) was used, the ionization efficiency of the analyte was higher and the peak type looked pretty; while when the mobile phase composition (ammonium formate [A] and acetonitrile [B]) was used, the ionization efficiency of the analyte was lower than the former and the peak type looked not well. Besides, the baseline was instability. Therefore, the mobile phase composition (ammonium formate [A] and methanol [B]) was selected.

Second, the concentration of ammonium formate was investigated. Ammonium formates in concentrations ranging from 5 mmol/L to 30 mmol/L were studied; it was found that the ionization efficiency of the analyte and the peak area were declined with the concentration of ammonium formate increased. Hence, the 5 mmol/L ammonium formate was determined to be used as our mobile phase (A).

Third, review previous studies; in order to obtain the most optimized mobile phase elution program, the following mobile phase ratio: (1) A:B = 30:70; (2) A:B = 27:73; (3) A:B = 35:65; (4) A:B = 40:60 and the corresponding gradient elution have been tried. The results showed that the peak type of A:B = 35:65 was the best, the ionization efficiency of A:B = 35:65 was the highest, the baseline of A:B = 35:65 was the most stable, and the retention time and peak area of A:B = 35:65 were the most stable. Ultimately, the methanol-5 mmol/L ammonium formate (65:35, v/v) with a desired separation at a flow rate of 0.3 mL/min was determined. The gradient program was as follows: 0-3 min, 65% B, 3.01 min, 65%-50% B, 3.02-5 min, 50% B, 5.01 min, 50%-15% B,5.02-7 min, 65% B.

About periplocin

The periplocin was also analyzed. At first, when we analyzed the standard solution of periplocin, the result was good. However, when we added the standard solution into the human blank blood, a high supererogatory peak was found near the same retention time; the same situation also appeared in the human blank blood, which seriously interfered the experimental results. The negative results about periplocin were shown in [Figure 2].
Figure 2: Total ion current chromatograms of standard solution (a), spiked blood (b), and blank blood (c). Standard solution with the 0.1 μg/mL internal standard, 0.5 μg/mL periplocin, and 0.2 μg/mL periplocymarin, blood sample spiked with the 0.1 μg/mL internal standard, 0.5 μg/mL periplocin and 0.2 μg/mL periplocymarin and blood sample. The retention times of periplocin, periplocymarin, and the internal standard were 1.235, 1.656, and 4.092 min, respectively, in standard solution (a). Serious endogenous interference was observed during the determination of periplocin

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Nevertheless, this phenomenon was not observed in rat blank blood according to relevant literature.[10],[11],[12],[14] It indicated that there was some compound in human body which was similar to periplocin. The specificity of the method to determine periplocin in human blood was too poor to analyze. According to report,[9] periplocin entered the body through oral administration which was decomposed by intestinal flora and quickly metabolized into periplocymarin. Furthermore, the periplocymarin also has been used for treatment of heart failure. Therefore, this method is mainly for the determination of periplocymarin in human blood and urine.

Validation of the optimized method

The optimized conditions were used to validate the method for the qualitative and quantitative analyses of periplocymarin.

For the qualitative analysis of periplocymarin, the selectivity of the method and the characteristic ion fragmentation were investigated in this study. The specificity of the method was assessed for the endogenous interference by comparing chromatograms of three different blank blood and blank urine samples; three blank blood and blank urine samples spiked with periplocymarin and IS, respectively. The total ion chromatogram of blank blood and spiked blood [Figure 3] reflected that no significant peaks were found at the retention time of periplocymarin or IS, which were detected at 3.857 and 2.502min; the total ion chromatogram of blank urine, spiked urine [Figure 4] reflected that no significant peaks were found at the retention time of periplocymarin or IS, which were detected at 4.061 and 2.663min. They were shown that there was no endogenous interference observed during the determination of periplocymarin from human blood and urine, and the separation efficiency of the periplocymarin and IS were great.
Figure 3: Total ion current chromatograms of (a) blank blood and (b) spiked blood. Blood sample spiked with the 0.1 μg/mL internal standard and 0.8 μg/mL periplocymarin. The retention times of periplocymarin and the internal standard were 3.857 and 2.502 min, respectively. No endogenous interference was observed during the determination of periplocymarin

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Figure 4: Total ion current chromatograms of (a) blank urine and (b) spiked urine. Urine sample spiked with the 0.1 μg/mL internal standard and 0.8 μg/mL periplocymarin. The retention times of periplocymarin and the internal standard were 4.061 and 2.663 min, respectively. No endogenous interference was observed during the determination of periplocymarin

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To determine the linearity of the method, the calibration standard solutions were prepared by spiking the blank human blood and urine (100 μL) with an appropriate amount of the working solutions yielding final concentrations of 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, and 1 μg/mL of periplocymarin and 100 ng/mL of digoxin-d3 (IS), respectively. The calibration curves were measured using the ratio of the peak areas of the target ion chosen for periplocymarin and those of the internal standard. Two calibration curves were constructed in the range 0.01-1 μg/mL. The regression equation of two curves and correlations coefficients (r2) were calculated as follows: Y = 3.3442X + 0.0265 (r2 = 0.9998, n = 7) (human blood), Y = 2.7594X + 0.0345 (r2 = 0.9996, n = 7) (human urine). They showed good linear relationship.

The limit of detection (LOD) of the developed method was determined by the analytes of the chromatographic extracts of the aqueous solution spiked with decreasing amounts of the analytes until a signal-to-noise (S/N) ratio of 3:1 was reached[17] The limit of quantification (LOQ) was estimated from the analysis as the concentration of an analyte giving an S/N ratio of 10:1. For periplocymarin in human blood and urine, the LOD and LOQ were all estimated to be 0.005 and 0.01 μg/ml, respectively.

The precision and accuracy of the method were expressed as the relative standard deviation (RSD) by treating three quality control samples at low, medium, and high concentrations (0.02, 0.1, and 0.8 μg/mL) of six replicates, and they were calculated using the calibration curves.[18] All replicates samples were tested under repeatable conditions (same analyst, same concentration, same preprocessing, same instrument, and same materials). The interday precision was determined by analyzing the six same-day replicates of blood samples and urine spiked at three levels. The intraday precision was processed by analysis of the samples on three consecutive days of six replicates. The accuracy was determined by the ratio of the measured concentration to the target concentration and the RSD at three levels of six replicates. The method in the human blood showed good intraday and interday precision with RSD values between 0.27%-3.34% and 0.32%-8.72%, respectively; the accuracy was 98.1%-99.5%, they are presented in [Table 2]. The method in the human urine showed good intraday and interday precision with RSD values between 0.56%-6.84% and 0.66%-8.59%, respectively; the accuracy was 96.2%-98%, they are presented in [Table 3]. An acceptable precision with RSD values of 20% at the lowest calibration point and 15% for higher concentrations is usually acceptable.[17],[18] The summary of accuracy and precision data indicated that this method was accurate and reproducible for the determination of periplocymarin in human blood and urine.
Table 2: The accuracy, precision, recovery, and matrix effects of the method in human blood (%)

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Table 3: The accuracy, precision, recovery, and matrix effects of the method in human urine (%)

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The extraction recoveries of periplocymarin at 0.02, 0.1, and 0.8 μg/mL levels were measured by comparing peak areas of periplocymarin in extracted samples with those in postextracted spiked samples. The recoveries of this analytical method in the human blood ranged from 97.1% to 98.3%, as shown in [Table 2], and the recoveries of this analytical method in the human urine ranged from 91.9% to 94.1%, as shown in [Table 3]. It can be seen that the extraction efficiency of the method was excellent, which can meet the requirements of forensic toxicology analysis.

The matrix effects of human blood and urine were obtained by the ratio of the peak areas of samples spiked postextraction to those in nonextracted samples at equivalent concentrations using six replicates. The matrix effects of human blood were from 95.3 ± 2.37% to 97.8 ± 1.88%, as shown in [Table 2], and the matrix effects of human urine were from 95.2 ± 1.70% to 96.8 ± 2.99%, as shown in [Table 3].


  Conclusion Top


The established LC-MS/MS method with a convenient extraction procedure for the qualitative and quantitative determination of periplocymarin in human blood and urine samples was simple, sensitive, and reliable. By methodological validation, the selectivity, linearity, accuracy, precision, extraction recovery, and matrix effect of periplocymarin could meet the requirements of forensic toxicological analysis and The method could provide some suggestions for the treatment of clinical periplocymarin poisoning cases.

Financial support and sponsorship

This study was financially supported by the Project of the National Natural Sciences Foundation of China (81373239).

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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