Original Article 607
UPLC-MS/MS Determination of Phentolamine in Human Plasma and its Application to a Pharmacokinetic Study
Affiliations
Key words
▶ phentolamine ● ▶ UPLC-MS/MS ● ▶ human plasma ● ▶ pharmacokinetic ●
X. Kan1, 4, S.-l. Zheng2, C.-y. Zhou3 1
Wenzhou People’s Hospital, Wenzhou, China The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China 3 The Third Affiliated Hospital of Wenzhou Medical University, Wenzhou, China 4 Yueqing Third People’s Hospital, Wenzhou, China 2
Abstract
▼
A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine phentolamine in human plasma. Sample preparation was accomplished through a simple liquid-liquid extraction with ethyl acetate. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column using an isocratic mobile phase system composed of acetonitrile and 1 % formic acid in water (33:67, v/v) at a flow rate of 0.45 mL/min. Mass spectrometric analysis was performed using a QTrap5500 mass spec-
Introduction
▼
received 21.12.2013 accepted 06.01.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1364002 Published online: January 22, 2014 Drug Res 2014; 64: 607–612 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence C.-y. Zhou The Third Affiliated Hospital of Wenzhou Medical University Wan Song Road 108 Rui’an Wenzhou 325000 China Tel.: + 86/577/88879 689 Fax: + 86/577/88879 698 [email protected]
Phentolamine is a nonselective, α-adrenergic blocking agent that has been available in the United States since 1952 for the clinical reversal of accidental extravasation of catecholamines during intravenous administration and for the diagnosis of pheochromocytoma [1–3]. The primary action of phentolamine is vasodilation [4, 5]. Clinical trials have evaluated the use of phentolamine with patients undergoing routine nonsurgical, operative, or periodontal procedures, implant placement, and in asymptomatic endodontic patients [6–8]. These well-controlled studies have shown that phentolamine statistically reduces the time of soft tissue numbness when compared with a sham injection. Side effects have been reported as minimal and similar to a sham injection [8]. In addition, it found that recovery of subjects’ perceptions of altered function, sensation, appearance, and actual function (speaking, smiling, drinking, and drooling) was quicker with phentolamine than with a sham injection [2]. Hence, it is quite essential to develop a simple and sensitive method to monitor phentolamine for obtaining optimum therapeutic concentrations in human blood.
trometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 282.1 → 212.0 and m/z 237.1 → 194.2 were used to quantify for phentolamine and carbamazepine (internal standard, IS), respectively. The linearity of this method was found to be within the concentration range of 0.5–100.0 ng/mL with a lower limit of quantification of 0.5 ng/mL. Only 1.0 min was needed for an analytical run. This fully validated method was successfully applied to the pharmacokinetic study after oral administration of 60 mg phentolamine to 20 Chinese healthy male volunteers.
Several analytical methods have been developed for the determination of phentolamine individually or in combination with other drugs including high performance liquid chromatography (HPLC) [9–12], HPLC tandem mass spectrometer (HPLCMS/MS) [13–15], thin-layerchromatography [16] and chemiluminescence [17, 18]. However, these methods have experienced some shortcomings that limited their applications to high sample throughput or pharmacokinetic studies in human. For example, several methods based on HPLC have long analytical run time more than 4 min. The sensitivity is relatively low and even large amount of plasma have been used. Moreover, no publication has described the quantitative analysis of phentolamine using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Therefore, a new simple and rapid method for measurement of phentolamine concentration in human plasma sample is still desirable. UPLC-MS/MS has been evaluated as a faster and more efficient analytical tool compared with current chromatography [19]. In the present study, we developed a UPLC-MS/MS method for the determination of phentolamine using carbamazepine as an internal standard (IS). This new
Kan X et al. Determination of Phentolamine … Drug Res 2014; 64: 607–612
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Authors
608 Original Article
method has been fully validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. It has been successfully applied in a pharmacokinetic study conducted in Chinese healthy male volunteers.
Materials and Methods
▼
Chemicals and reagents Phentolamine and carbamazepine (internal standard, IS) were obtained from Sigma (St. Louis, MO, USA). LC-grade acetonitrile, ethyl acetate, sodium hydroxide and sodium dihydrogen orthophosphate (NaH2PO4) were purchased from the Beijing Chemical Reagents Company (Beijing, China). HPLC grade water was obtained using a Milli Q system (Millipore, Bedford, USA). Blank human plasma used in this study was supplied by The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China).
Standard solutions, calibration standards and quality control (QC) sample The stock solution of phentolamine that was used to make the calibration standards and quality control (QC) samples was prepared by dissolving 10 mg in 10 mL methanol to obtain a concentration of 1.00 mg/mL. The stock solution was further diluted with methanol to obtain working solutions at several concentration levels. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank human plasma. Final concentrations of the calibration standards were 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0 and 100.0 ng/mL for phentolamine in human plasma. The concentrations of QC samples in plasma were 1.0, 10.0, 80.0 ng/mL for phentolamine. IS stock solution was made at an initial concentration of 1 mg/mL. The IS working solution (1 000 ng/mL) was made from the stock solution using methanol for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at 4 °C.
Sample preparation UPLC-MS/MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) and inline 0.2 μm stainless steel frit filter (Waters Corp., Milford, USA). The mobile phase consisted of acetonitrile and 1 % formic acid in water (33:67, v/v). The flow rate was 0.45 mL/min. The overall run time was 1.0 min. An AB Sciex QTRAP 5500 triple quadruple mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Canada) was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution (0.7 amu) in the mass analyzers. The dwell time was set to 250 ms for each MRM transition. The MRM transitions were m/z 282.1 → 212.0 and m/z 237.1 → 194.2 for phen▶ Fig. 1). After optimization, the tolamine and IS, respectively (● source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 60 psig; ion spray voltage, 3.5 kV; and temperature, 350 °C. Data acquiring and processing were performed using analyst software (version 1.5, AB Sciex).
Kan X et al. Determination of Phentolamine … Drug Res 2014; 64: 607–612
Plasma samples were thawed to room temperature and vortexed thoroughly before use. To 500 μL plasma sample, 50 μL IS (1 000 ng/mL) and 500 μL 0.1 M sodium hydroxide solution were added in a 10 mL test tube. After adding 5.0 mL ethyl acetate, the test tubes were vortex-mixed thoroughly for 2 min. The supernatant of organic phase was separated and evaporated to dryness at 50 °C under a gentle stream of nitrogen. The residue was dissolved in 150 μL of mobile phase and centrifuged at 12 000 rpm for 5 min. Clear supernatant was collected and only 10 μL injected into the UPLC-MS/MS system for analysis.
Method validation Before using this method to determinate phentolamine in human plasma, the method was fully validated for specificity, linearity, precision, accuracy, recovery, matrix effect and stability according the United States Food and Drug Administration (US FDA) bioanalytical method validation guidances [20]. Specificity was determined by analysis of blank human plasma samples from 6 different volunteers, every blank sample was handled by the procedure described in “Sample preparation”
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Fig. 1 The chemical structures and daughter scan ion spectra of phentolamine and IS in the present study: a phentolamine; b carbamazepine (IS).
Original Article 609
Application to a pharmacokinetic study The present method was applied to a pharmacokinetic study after an oral administration of 60 mg phentolamine to Chinese male volunteers. The clinical protocol was approved by Medical Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University prior to the study. 20 volunteers were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. The volunteers who submitted the agreements to attend this project were medically examined for the pharmacokinetics study of phentolamine. The subjects were required to abstain from taking any other drug for 7 days prior to the start of test. They were also demanded not to smoke or drink alcohol for 24 h before the beginning of the study until its end. All volunteers were received an oral dose of 60 mg phentolamine with 200 mL water. Blood samples (3 mL) were collected into heparinized tubes at 5, 15, 30, 45 min, 1, 1.5, 2, 3, 4, 6, 8, and 12 h after oral administration.
Blood samples were centrifuged at 4 000 × g for 10 min and the plasma was separated and kept frozen at − 80 °C until analysis.
Data analysis Plasma concentration vs. time profiles were analyzed using DAS software (Version 2.0, Medical University of Wenzhou, China) to estimate the type of compartment model and pharmacokinetic parameters. Data were expressed as mean ± SD.
Results and Discussion
▼
Method development and optimization Analysis method was set up by optimizing UPLC and MS/MS conditions to obtain the best possible sensitivity. The analytes were analyzed firstly using MS by syringe infusion of individual standard solution and they were all more efficiently ionized in ESI positive mode than in negative mode. ESI positive mode was therefore employed. Parameters such as ESI source temperature, capillary and cone voltage, flow rate of desolvation gas and cone gas were optimized to obtain highest intensity of protonated molecules of analytes. The collision energy of collision-induced decomposition (CID) was optimized for maximum response of the fragmentation of analytes. MRM was used to monitor precursor ion and production, which could reduce interference and enhance selectivity. Sample preparation is a key step for accurate and reliable UPLCMS/MS assays. 2 kinds of sample treatment procedures were evaluated, including protein precipitation and liquid-liquid extraction. Liquid-liquid extraction could prepare purified and concentrated samples and improve the sensitivity and robustness of the assay. Protein precipitation was often used for the preparation of biological samples with the advantages of saving time and simplicity. In this work, 2 methods, including a protein precipitation procedure and ethyl acetate extraction, were investigated and compared. Because of its little interfering endogenous substances and the high recovery, liquid-liquid extraction was employed as the extraction method. Chromatographic conditions were optimized to achieve good sensitivity and peak shape for phentolamine and IS, as well as a short run time. We found a sharp peak for phentolamine and IS with good sensitivity could be achieved using a mobile phase consisting of acetonitrile and 1 % formic acid in water (33:67, v/v) on an Acquity UPLC BEH C18 column. After optimization, the retention times for phentolamine and IS were 0.63 min and 0.49 min, respectively. The overall run time was only 1.0 min. The advantage of this method is that a relatively larger number of samples can be analyzed in a short time, thus increasing output.
Specificity UPLC-MS/MS chromatogram of the analytes in human plasma ▶ Fig. 2. The retention times of phensamples were shown in ● tolamine and IS are 0.63 and 0.49 min, respectively. Compared with chromatogram of blank blood sample, no interference of endogenous peaks was observed.
Linearity and sensitivity The peak area ratios of phentolamine/IS vs. the nominal concentrations of phentolamine showed a good linear relationship over the concentration ranges of 0.5–100.0 ng/mL in human plasma. A typical regression equation for the calibration curve resulted Kan X et al. Determination of Phentolamine … Drug Res 2014; 64: 607–612
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and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. To evaluate the linearity, calibration standards of 9 concentrations of phentolamine (0.5–100.0 ng/mL) were separately extracted and assayed on 3 separate days. The linearity for phentolamine was investigated by weighted (1/x2) least-squares linear regression of peak area ratios against concentrations. The sensitivity of the method was determined by quantifying the lower limit of quantification (LLOQ). The LLOQ was defined as the lowest acceptable point in the calibration curve which were determined at an acceptable precision and accuracy. The precision and accuracy of the method were assessed by determination of QC samples in plasma at different concentrations (1.0, 10.0, 80.0 ng/mL) on 3 separate days. Precision was expressed as % relative standard deviation (RSD) and accuracy was expressed as % relative error (RE) between the measured and nominal value. The precision for QC samples was within 15 %, and accuracy between −15 and 15 %. Extraction recovery experiments which showed an ability to extract the analyte from the test biological samples, were evaluated by comparing the peak areas obtained from extracted QC samples with non-processed standard solutions at 3 concentrations at the same concentration. Recovery of IS was determined at the working concentration (1 000 ng/mL) similarly. To determine the matrix effect, 6 different blank human samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect was evaluated at 3 QC levels by comparing the peak areas of analytes obtained from plasma samples spiked with analytes after extraction to those of the pure standard solutions at the same concentrations. The matrix effect of IS was evaluated at the working concentration (1 000 ng/mL) in the same manner. To ensure the reliability of the results, stability assay comprising freeze-thaw stability, short-term and long-term stability were carried out. In the protocol, the short-term stability was determined after the exposure of the spiked samples at room temperature for 4 h, and the ready-to-inject samples (after extraction) in the autosampler at 4 °C for 24 h. The freeze-thaw stability was evaluated after 3 complete freeze-thaw cycles ( − 80 to 25 °C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at − 80 °C for 28 days. Samples were considered to be stable if assay values were within the acceptable limits of accuracy (RE % ≤ ± 15 %) and precision (RSD % ≤ 15 %).
610 Original Article
in an equation of the line of: y = (101.341x + 0.055, r = 0.9998), where y represents the peak area ratios of phentolamine to the IS and x represents plasma concentrations of analyte. The LLOQ in human plasma was 0.5 ng/mL with the RSD and RE of 7.6 and 8.6 %, respectively.
The matrix effect in human plasma were all between 97.33 % to ▶ Table 2). The 103.83 % for phentolamine at different QC levels (● matrix effect for IS (1 000 ng/mL) was 96.87 %. No apparent matrix effect was found to affect the determination of phentolamine and IS in plasma. As a result, the matrix effect from plasma was negligible in this method.
Precision and accuracy
Recovery and matrix effect The recovery was calculated by comparing the mean peak areas of the analyte spiked before extraction divided by the areas of analytes samples spiked after extraction and multiplied by 100. ▶ Table 2. The recovery in plasma ranged Results are shown in ● from 90.67 % to 96.83 % for phentolamine. The recovery of IS (1 000 ng/mL) in plasma was 90.48 %.
Stability Stability tests were performed at the low, medium and high QC samples with 5 determinations for each under different storage conditions. The RSDs of the mean test responses were within 15 % in all stability tests. ▶ Table 3 shows the stability data for phentolamine in plasma ● under different storage and temperature conditions. There was no effect on the quantitation for plasma samples kept at room temperature for 4 h and at 4 °C for 24 h. No significant degradation was observed when samples of phentolamine were taken through 3 freeze ( − 80 °C) – thaw (room temperature) cycles. As a result, phentolamine in samples were stable at − 80 °C for 28 days.
Fig. 2 Representative chromatograms of phentolamine and IS in human plasma samples. a a blank plasma sample; b a blank plasma sample spiked with phentolamine and IS; c a plasma sample from a person 1.0 h after an oral administration 60 mg.
Analytes
Concentration
Intra-day precision
added (ng/mL)
Mean ± SD
Phentolamine
1.0 10.0 80.0
0.96 ± 0.06 9.61 ± 1.08 74.67 ± 4.37
Analytes
Concentration
Recovery ( %)
added (ng/mL)
Mean ± SD
RSD ( %)
Mean ± SD
RSD ( %)
1.0 10.0 80.0 1 000
93.67 ± 6.25 90.67 ± 3.93 96.83 ± 3.49 90.48 ± 3.84
6.67 4.33 3.60 4.24
97.33 ± 5.05 103.83 ± 7.00 99.17 ± 6.31 96.87 ± 5.45
5.18 6.74 6.36 5.63
Phentolamine IS
Inter-day precision
RSD ( %)
RE ( %)
Mean ± SD
RSD ( %)
RE ( %)
6.74 11.26 5.85
− 3.98 − 3.87 − 6.67
1.06 ± 0.11 8.99 ± 1.04 78.67 ± 4.68
10.39 11.56 5.94
5.88 − 10.15 − 1.67
Matrix effect ( %)
Kan X et al. Determination of Phentolamine … Drug Res 2014; 64: 607–612
Table 1 Precision and accuracy of method for the determination of phentolamine in human plasma (n = 6).
Table 2 Recovery and matrix effect of phentolamine and internal standards (n = 6).
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The intra- and inter-day precision and accuracy of the method were determined from the analysis of QC samples at 3 different concentrations for each biological matrix. The results are sum▶ Table 1. The method was reliable and reproducible marized in ● since RSD % was below 15 % and RE % was between − 10.15 % and 5.88 % for all the investigated concentrations of phentolamine in human plasma.
Original Article 611
Table 3 Stability results of phentolamine in human plasma in different conditions (n = 5). Analytes
Concentration added
Ambient, 4 h Mean ± SD
(ng/mL) Phentolamine
1.0 10.0 80.0
0.93 ± 0.05 9.82 ± 0.85 89.33 ± 6.25
4 °C, 24 h
RSD
RE
( %) 5.92 8.63 7.00
Mean ± SD
Three freeze-thaw
RSD
RE
( %)
( %)
( %)
− 7.17 0.94 ± 0.10 − 1.80 9.47 ± 0.99 11.67 85.33 ± 6.44
10.23 10.45 7.55
− 6.33 − 5.32 6.67
Mean ± SD 0.90 ± 0.06 9.67 ± 0.60 72.17 ± 2.64
− 80 °C, 28 days
RSD
RE
Mean ± SD
RSD
RE
( %)
( %)
( %)
( %)
6.62 6.20 3.66
− 10.17 1.03 ± 0.09 − 3.33 10.05 ± 0.61 − 9.79 75.83 ± 3.19
8.51 6.06 4.20
3.03 0.50 − 5.21
our knowledge, this is the first report of the determination of phentolamine level in human plasma using an UPLC-MS/MS method. The method affords the sensitivity, accuracy and precision needed for quantitative measurements of phentolamine in human plasma. It was also successfully applied in a pharmacokinetic study.
Conflict of Interest The authors report no conflicts of interest.
References
Fig. 3 Plasma concentration vs. time curves of phentolamine after a single oral administration 60 mg to 20 Chinese healthy male volunteers.
Table 4 Pharmacokinetic parameters of phentolamine after oral administration 60 mg to 20 Chinese healthy male volunteers. Parameter
Phentolamine
t1/2 (h) Cmax (ng/mL) Tmax (h) AUC0→12 (ng/mL ∙ h) AUC0→∞ (ng/mL ∙ h)
3.32 ± 0.85 67.05 ± 14.34 0.74 ± 0.22 182.03 ± 34.59 197.59 ± 36.87
Application of the method in a pharmacokinetic study The method described above was successfully applied to determine the concentration of phentolamine in human plasma. Ethical approval was given by the medical ethics committee of The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). After an oral administration of 60 mg phentolamine tablets, the main pharmacokinetic parameters of phentolamine for 20 volunteers were estimated. The mean plasma concentration-time curve of phentolamine was dis▶ Fig. 3, and the main pharmacokinetic parameters of played in ● ▶ Table 4. phentolamine were calculated and are summarized in ● Compared with recently published papers describing the pharmacokinetic profiles of phentolamine in healthy volunteers, the pharmacokinetic parameters of phentolamine obtained in the study were generally similar [15].
Conclusions
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1 Moore PA, Hersh EV, Papas AS et al. Pharmacokinetics of lidocaine with epinephrine following local anesthesia reversal with phentolamine mesylate. Anesth Prog 2008; 55: 40–48 2 Hersh EV, Moore PA, Papas AS et al. Reversal of soft-tissue local anesthesia with phentolamine mesylate in adolescents and adults. J Am Dent Assoc 2008; 139: 1080–1093 3 Elmore S, Nusstein J, Drum M et al. Reversal of Pulpal and Soft Tissue Anesthesia by Using Phentolamine: A Prospective Randomized, Single-blind Study. J Endod 2013; 39: 429–434 4 Tavares M, Goodson JM, Studen-Pavlovich D et al. Reversal of soft-tissue local anesthesia with phentolamine mesylate in pediatric patients. J Am Dent Assoc 2008; 139: 1095–1104 5 Moradkhan R, Spitnale B, McQuillan P et al. Hypoxia-induced vasodilation and effects of regional phentolamine in awake patients with sleep apnea. J Appl Physiol 2010; 108: 1234–1240 6 Laviola M, McGavin SK, Freer GA et al. Randomized study of phentolamine mesylate for reversal of local anesthesia. J Dent Res 2008; 87: 635–639 7 Froum SJ, Froum SH, Malamed SF. The use of phentolamine mesylate to evaluate mandibular nerve damage following implant placement. Compend Contin Educ Dent 2010; 31: 520, 522–528 8 Fowler S, Nusstein J, Drum M et al. Reversal of soft-tissue anesthesia in asymptomatic endodontic patients: a preliminary, prospective, randomized, single-blind study. J Endod 2011; 37: 1353–1358 9 Webster GK, Lemmer RR, Greenwald S. Rapid analysis of phentolamine by high-performance liquid chromatography. J Chromatogr Sci 2003; 41: 57–62 10 Kerger BD, James RC, Roberts SM. An assay for phentolamine using high performance liquid chromatography with electrochemical detection. Anal Biochem 1988; 170: 145–151 11 Godbillon J, Carnis G. Determination of the major metabolite of phentolamine in human plasma and urine by high-performance liquid chromatography. J Chromatogr 1981; 222: 461–466 12 de Bros F, Wolshin EM. Determination of phentolamine in blood and urine by high performance liquid chromatography. Anal Chem 1978; 50: 521–525 13 Wu L, Liu J, Zhang Y et al. Development of a HPLC/MS/MS method for simultaneous determination of tinidazole, dyclonine and chlorhexidine in rat plasma and its application in the pharmacokinetic research of a film-forming solution. J Pharm Biomed Anal 2012; 62: 224–227 14 Wu JT. The development of a staggered parallel separation liquid chromatography/tandem mass spectrometry system with on-line extraction for high-throughout screening of drug candidates in biological fluids. Rapid Commun Mass Spectrom 2001; 15: 73–81 15 Silva LF, Moraes MO, Santana GS et al. Phentolamine bioequivalence study. Int J Clin Pharmacol Ther 2004; 42: 43–49
A UPLC-MS/MS method for the determination of phentolamine in human plasma was developed and validated. To the best of Kan X et al. Determination of Phentolamine … Drug Res 2014; 64: 607–612
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16 Mikami E, Ohno T, Matsumoto H. Simultaneous identification/determination system for phentolamine and sildenafil as adulterants in soft drinks advertising roborant nutrition. Forensic Sci Int 2002; 130: 140–146 17 Xing LL, Tang YH, Wang ZC et al. Sensitive chemiluminescence determination of phentolamine mesylate and phenoxybenzamine hydrochloride based on K3Fe(CN)(6)-H2O2-fluorescein. Journal of Luminescence 2013; 137: 162–167 18 Huang Y, Liu W. Flow-injection chemiluminescence determination of phentolamine based on its enhancing effect on the luminol-potassium ferricyanide system. J Pharm Biomed Anal 2005; 38: 537–542
Kan X et al. Determination of Phentolamine … Drug Res 2014; 64: 607–612
UPLC-MS/MS Determination of Phentolamine in Human Plasma and its Application to a Pharmacokinetic Study
Affiliations
Key words
▶ phentolamine ● ▶ UPLC-MS/MS ● ▶ human plasma ● ▶ pharmacokinetic ●
X. Kan1, 4, S.-l. Zheng2, C.-y. Zhou3 1
Wenzhou People’s Hospital, Wenzhou, China The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China 3 The Third Affiliated Hospital of Wenzhou Medical University, Wenzhou, China 4 Yueqing Third People’s Hospital, Wenzhou, China 2
Abstract
▼
A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine phentolamine in human plasma. Sample preparation was accomplished through a simple liquid-liquid extraction with ethyl acetate. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column using an isocratic mobile phase system composed of acetonitrile and 1 % formic acid in water (33:67, v/v) at a flow rate of 0.45 mL/min. Mass spectrometric analysis was performed using a QTrap5500 mass spec-
Introduction
▼
received 21.12.2013 accepted 06.01.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1364002 Published online: January 22, 2014 Drug Res 2014; 64: 607–612 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence C.-y. Zhou The Third Affiliated Hospital of Wenzhou Medical University Wan Song Road 108 Rui’an Wenzhou 325000 China Tel.: + 86/577/88879 689 Fax: + 86/577/88879 698 [email protected]
Phentolamine is a nonselective, α-adrenergic blocking agent that has been available in the United States since 1952 for the clinical reversal of accidental extravasation of catecholamines during intravenous administration and for the diagnosis of pheochromocytoma [1–3]. The primary action of phentolamine is vasodilation [4, 5]. Clinical trials have evaluated the use of phentolamine with patients undergoing routine nonsurgical, operative, or periodontal procedures, implant placement, and in asymptomatic endodontic patients [6–8]. These well-controlled studies have shown that phentolamine statistically reduces the time of soft tissue numbness when compared with a sham injection. Side effects have been reported as minimal and similar to a sham injection [8]. In addition, it found that recovery of subjects’ perceptions of altered function, sensation, appearance, and actual function (speaking, smiling, drinking, and drooling) was quicker with phentolamine than with a sham injection [2]. Hence, it is quite essential to develop a simple and sensitive method to monitor phentolamine for obtaining optimum therapeutic concentrations in human blood.
trometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 282.1 → 212.0 and m/z 237.1 → 194.2 were used to quantify for phentolamine and carbamazepine (internal standard, IS), respectively. The linearity of this method was found to be within the concentration range of 0.5–100.0 ng/mL with a lower limit of quantification of 0.5 ng/mL. Only 1.0 min was needed for an analytical run. This fully validated method was successfully applied to the pharmacokinetic study after oral administration of 60 mg phentolamine to 20 Chinese healthy male volunteers.
Several analytical methods have been developed for the determination of phentolamine individually or in combination with other drugs including high performance liquid chromatography (HPLC) [9–12], HPLC tandem mass spectrometer (HPLCMS/MS) [13–15], thin-layerchromatography [16] and chemiluminescence [17, 18]. However, these methods have experienced some shortcomings that limited their applications to high sample throughput or pharmacokinetic studies in human. For example, several methods based on HPLC have long analytical run time more than 4 min. The sensitivity is relatively low and even large amount of plasma have been used. Moreover, no publication has described the quantitative analysis of phentolamine using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Therefore, a new simple and rapid method for measurement of phentolamine concentration in human plasma sample is still desirable. UPLC-MS/MS has been evaluated as a faster and more efficient analytical tool compared with current chromatography [19]. In the present study, we developed a UPLC-MS/MS method for the determination of phentolamine using carbamazepine as an internal standard (IS). This new
Kan X et al. Determination of Phentolamine … Drug Res 2014; 64: 607–612
Downloaded by: Harvard University Library. Copyrighted material.
Authors
608 Original Article
method has been fully validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. It has been successfully applied in a pharmacokinetic study conducted in Chinese healthy male volunteers.
Materials and Methods
▼
Chemicals and reagents Phentolamine and carbamazepine (internal standard, IS) were obtained from Sigma (St. Louis, MO, USA). LC-grade acetonitrile, ethyl acetate, sodium hydroxide and sodium dihydrogen orthophosphate (NaH2PO4) were purchased from the Beijing Chemical Reagents Company (Beijing, China). HPLC grade water was obtained using a Milli Q system (Millipore, Bedford, USA). Blank human plasma used in this study was supplied by The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China).
Standard solutions, calibration standards and quality control (QC) sample The stock solution of phentolamine that was used to make the calibration standards and quality control (QC) samples was prepared by dissolving 10 mg in 10 mL methanol to obtain a concentration of 1.00 mg/mL. The stock solution was further diluted with methanol to obtain working solutions at several concentration levels. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank human plasma. Final concentrations of the calibration standards were 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0 and 100.0 ng/mL for phentolamine in human plasma. The concentrations of QC samples in plasma were 1.0, 10.0, 80.0 ng/mL for phentolamine. IS stock solution was made at an initial concentration of 1 mg/mL. The IS working solution (1 000 ng/mL) was made from the stock solution using methanol for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at 4 °C.
Sample preparation UPLC-MS/MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) and inline 0.2 μm stainless steel frit filter (Waters Corp., Milford, USA). The mobile phase consisted of acetonitrile and 1 % formic acid in water (33:67, v/v). The flow rate was 0.45 mL/min. The overall run time was 1.0 min. An AB Sciex QTRAP 5500 triple quadruple mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Canada) was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution (0.7 amu) in the mass analyzers. The dwell time was set to 250 ms for each MRM transition. The MRM transitions were m/z 282.1 → 212.0 and m/z 237.1 → 194.2 for phen▶ Fig. 1). After optimization, the tolamine and IS, respectively (● source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 60 psig; ion spray voltage, 3.5 kV; and temperature, 350 °C. Data acquiring and processing were performed using analyst software (version 1.5, AB Sciex).
Kan X et al. Determination of Phentolamine … Drug Res 2014; 64: 607–612
Plasma samples were thawed to room temperature and vortexed thoroughly before use. To 500 μL plasma sample, 50 μL IS (1 000 ng/mL) and 500 μL 0.1 M sodium hydroxide solution were added in a 10 mL test tube. After adding 5.0 mL ethyl acetate, the test tubes were vortex-mixed thoroughly for 2 min. The supernatant of organic phase was separated and evaporated to dryness at 50 °C under a gentle stream of nitrogen. The residue was dissolved in 150 μL of mobile phase and centrifuged at 12 000 rpm for 5 min. Clear supernatant was collected and only 10 μL injected into the UPLC-MS/MS system for analysis.
Method validation Before using this method to determinate phentolamine in human plasma, the method was fully validated for specificity, linearity, precision, accuracy, recovery, matrix effect and stability according the United States Food and Drug Administration (US FDA) bioanalytical method validation guidances [20]. Specificity was determined by analysis of blank human plasma samples from 6 different volunteers, every blank sample was handled by the procedure described in “Sample preparation”
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Fig. 1 The chemical structures and daughter scan ion spectra of phentolamine and IS in the present study: a phentolamine; b carbamazepine (IS).
Original Article 609
Application to a pharmacokinetic study The present method was applied to a pharmacokinetic study after an oral administration of 60 mg phentolamine to Chinese male volunteers. The clinical protocol was approved by Medical Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University prior to the study. 20 volunteers were given written informed consent to participate in the study according to the principles of the Declaration of Helsinki. The volunteers who submitted the agreements to attend this project were medically examined for the pharmacokinetics study of phentolamine. The subjects were required to abstain from taking any other drug for 7 days prior to the start of test. They were also demanded not to smoke or drink alcohol for 24 h before the beginning of the study until its end. All volunteers were received an oral dose of 60 mg phentolamine with 200 mL water. Blood samples (3 mL) were collected into heparinized tubes at 5, 15, 30, 45 min, 1, 1.5, 2, 3, 4, 6, 8, and 12 h after oral administration.
Blood samples were centrifuged at 4 000 × g for 10 min and the plasma was separated and kept frozen at − 80 °C until analysis.
Data analysis Plasma concentration vs. time profiles were analyzed using DAS software (Version 2.0, Medical University of Wenzhou, China) to estimate the type of compartment model and pharmacokinetic parameters. Data were expressed as mean ± SD.
Results and Discussion
▼
Method development and optimization Analysis method was set up by optimizing UPLC and MS/MS conditions to obtain the best possible sensitivity. The analytes were analyzed firstly using MS by syringe infusion of individual standard solution and they were all more efficiently ionized in ESI positive mode than in negative mode. ESI positive mode was therefore employed. Parameters such as ESI source temperature, capillary and cone voltage, flow rate of desolvation gas and cone gas were optimized to obtain highest intensity of protonated molecules of analytes. The collision energy of collision-induced decomposition (CID) was optimized for maximum response of the fragmentation of analytes. MRM was used to monitor precursor ion and production, which could reduce interference and enhance selectivity. Sample preparation is a key step for accurate and reliable UPLCMS/MS assays. 2 kinds of sample treatment procedures were evaluated, including protein precipitation and liquid-liquid extraction. Liquid-liquid extraction could prepare purified and concentrated samples and improve the sensitivity and robustness of the assay. Protein precipitation was often used for the preparation of biological samples with the advantages of saving time and simplicity. In this work, 2 methods, including a protein precipitation procedure and ethyl acetate extraction, were investigated and compared. Because of its little interfering endogenous substances and the high recovery, liquid-liquid extraction was employed as the extraction method. Chromatographic conditions were optimized to achieve good sensitivity and peak shape for phentolamine and IS, as well as a short run time. We found a sharp peak for phentolamine and IS with good sensitivity could be achieved using a mobile phase consisting of acetonitrile and 1 % formic acid in water (33:67, v/v) on an Acquity UPLC BEH C18 column. After optimization, the retention times for phentolamine and IS were 0.63 min and 0.49 min, respectively. The overall run time was only 1.0 min. The advantage of this method is that a relatively larger number of samples can be analyzed in a short time, thus increasing output.
Specificity UPLC-MS/MS chromatogram of the analytes in human plasma ▶ Fig. 2. The retention times of phensamples were shown in ● tolamine and IS are 0.63 and 0.49 min, respectively. Compared with chromatogram of blank blood sample, no interference of endogenous peaks was observed.
Linearity and sensitivity The peak area ratios of phentolamine/IS vs. the nominal concentrations of phentolamine showed a good linear relationship over the concentration ranges of 0.5–100.0 ng/mL in human plasma. A typical regression equation for the calibration curve resulted Kan X et al. Determination of Phentolamine … Drug Res 2014; 64: 607–612
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and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. To evaluate the linearity, calibration standards of 9 concentrations of phentolamine (0.5–100.0 ng/mL) were separately extracted and assayed on 3 separate days. The linearity for phentolamine was investigated by weighted (1/x2) least-squares linear regression of peak area ratios against concentrations. The sensitivity of the method was determined by quantifying the lower limit of quantification (LLOQ). The LLOQ was defined as the lowest acceptable point in the calibration curve which were determined at an acceptable precision and accuracy. The precision and accuracy of the method were assessed by determination of QC samples in plasma at different concentrations (1.0, 10.0, 80.0 ng/mL) on 3 separate days. Precision was expressed as % relative standard deviation (RSD) and accuracy was expressed as % relative error (RE) between the measured and nominal value. The precision for QC samples was within 15 %, and accuracy between −15 and 15 %. Extraction recovery experiments which showed an ability to extract the analyte from the test biological samples, were evaluated by comparing the peak areas obtained from extracted QC samples with non-processed standard solutions at 3 concentrations at the same concentration. Recovery of IS was determined at the working concentration (1 000 ng/mL) similarly. To determine the matrix effect, 6 different blank human samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect was evaluated at 3 QC levels by comparing the peak areas of analytes obtained from plasma samples spiked with analytes after extraction to those of the pure standard solutions at the same concentrations. The matrix effect of IS was evaluated at the working concentration (1 000 ng/mL) in the same manner. To ensure the reliability of the results, stability assay comprising freeze-thaw stability, short-term and long-term stability were carried out. In the protocol, the short-term stability was determined after the exposure of the spiked samples at room temperature for 4 h, and the ready-to-inject samples (after extraction) in the autosampler at 4 °C for 24 h. The freeze-thaw stability was evaluated after 3 complete freeze-thaw cycles ( − 80 to 25 °C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at − 80 °C for 28 days. Samples were considered to be stable if assay values were within the acceptable limits of accuracy (RE % ≤ ± 15 %) and precision (RSD % ≤ 15 %).
610 Original Article
in an equation of the line of: y = (101.341x + 0.055, r = 0.9998), where y represents the peak area ratios of phentolamine to the IS and x represents plasma concentrations of analyte. The LLOQ in human plasma was 0.5 ng/mL with the RSD and RE of 7.6 and 8.6 %, respectively.
The matrix effect in human plasma were all between 97.33 % to ▶ Table 2). The 103.83 % for phentolamine at different QC levels (● matrix effect for IS (1 000 ng/mL) was 96.87 %. No apparent matrix effect was found to affect the determination of phentolamine and IS in plasma. As a result, the matrix effect from plasma was negligible in this method.
Precision and accuracy
Recovery and matrix effect The recovery was calculated by comparing the mean peak areas of the analyte spiked before extraction divided by the areas of analytes samples spiked after extraction and multiplied by 100. ▶ Table 2. The recovery in plasma ranged Results are shown in ● from 90.67 % to 96.83 % for phentolamine. The recovery of IS (1 000 ng/mL) in plasma was 90.48 %.
Stability Stability tests were performed at the low, medium and high QC samples with 5 determinations for each under different storage conditions. The RSDs of the mean test responses were within 15 % in all stability tests. ▶ Table 3 shows the stability data for phentolamine in plasma ● under different storage and temperature conditions. There was no effect on the quantitation for plasma samples kept at room temperature for 4 h and at 4 °C for 24 h. No significant degradation was observed when samples of phentolamine were taken through 3 freeze ( − 80 °C) – thaw (room temperature) cycles. As a result, phentolamine in samples were stable at − 80 °C for 28 days.
Fig. 2 Representative chromatograms of phentolamine and IS in human plasma samples. a a blank plasma sample; b a blank plasma sample spiked with phentolamine and IS; c a plasma sample from a person 1.0 h after an oral administration 60 mg.
Analytes
Concentration
Intra-day precision
added (ng/mL)
Mean ± SD
Phentolamine
1.0 10.0 80.0
0.96 ± 0.06 9.61 ± 1.08 74.67 ± 4.37
Analytes
Concentration
Recovery ( %)
added (ng/mL)
Mean ± SD
RSD ( %)
Mean ± SD
RSD ( %)
1.0 10.0 80.0 1 000
93.67 ± 6.25 90.67 ± 3.93 96.83 ± 3.49 90.48 ± 3.84
6.67 4.33 3.60 4.24
97.33 ± 5.05 103.83 ± 7.00 99.17 ± 6.31 96.87 ± 5.45
5.18 6.74 6.36 5.63
Phentolamine IS
Inter-day precision
RSD ( %)
RE ( %)
Mean ± SD
RSD ( %)
RE ( %)
6.74 11.26 5.85
− 3.98 − 3.87 − 6.67
1.06 ± 0.11 8.99 ± 1.04 78.67 ± 4.68
10.39 11.56 5.94
5.88 − 10.15 − 1.67
Matrix effect ( %)
Kan X et al. Determination of Phentolamine … Drug Res 2014; 64: 607–612
Table 1 Precision and accuracy of method for the determination of phentolamine in human plasma (n = 6).
Table 2 Recovery and matrix effect of phentolamine and internal standards (n = 6).
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The intra- and inter-day precision and accuracy of the method were determined from the analysis of QC samples at 3 different concentrations for each biological matrix. The results are sum▶ Table 1. The method was reliable and reproducible marized in ● since RSD % was below 15 % and RE % was between − 10.15 % and 5.88 % for all the investigated concentrations of phentolamine in human plasma.
Original Article 611
Table 3 Stability results of phentolamine in human plasma in different conditions (n = 5). Analytes
Concentration added
Ambient, 4 h Mean ± SD
(ng/mL) Phentolamine
1.0 10.0 80.0
0.93 ± 0.05 9.82 ± 0.85 89.33 ± 6.25
4 °C, 24 h
RSD
RE
( %) 5.92 8.63 7.00
Mean ± SD
Three freeze-thaw
RSD
RE
( %)
( %)
( %)
− 7.17 0.94 ± 0.10 − 1.80 9.47 ± 0.99 11.67 85.33 ± 6.44
10.23 10.45 7.55
− 6.33 − 5.32 6.67
Mean ± SD 0.90 ± 0.06 9.67 ± 0.60 72.17 ± 2.64
− 80 °C, 28 days
RSD
RE
Mean ± SD
RSD
RE
( %)
( %)
( %)
( %)
6.62 6.20 3.66
− 10.17 1.03 ± 0.09 − 3.33 10.05 ± 0.61 − 9.79 75.83 ± 3.19
8.51 6.06 4.20
3.03 0.50 − 5.21
our knowledge, this is the first report of the determination of phentolamine level in human plasma using an UPLC-MS/MS method. The method affords the sensitivity, accuracy and precision needed for quantitative measurements of phentolamine in human plasma. It was also successfully applied in a pharmacokinetic study.
Conflict of Interest The authors report no conflicts of interest.
References
Fig. 3 Plasma concentration vs. time curves of phentolamine after a single oral administration 60 mg to 20 Chinese healthy male volunteers.
Table 4 Pharmacokinetic parameters of phentolamine after oral administration 60 mg to 20 Chinese healthy male volunteers. Parameter
Phentolamine
t1/2 (h) Cmax (ng/mL) Tmax (h) AUC0→12 (ng/mL ∙ h) AUC0→∞ (ng/mL ∙ h)
3.32 ± 0.85 67.05 ± 14.34 0.74 ± 0.22 182.03 ± 34.59 197.59 ± 36.87
Application of the method in a pharmacokinetic study The method described above was successfully applied to determine the concentration of phentolamine in human plasma. Ethical approval was given by the medical ethics committee of The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). After an oral administration of 60 mg phentolamine tablets, the main pharmacokinetic parameters of phentolamine for 20 volunteers were estimated. The mean plasma concentration-time curve of phentolamine was dis▶ Fig. 3, and the main pharmacokinetic parameters of played in ● ▶ Table 4. phentolamine were calculated and are summarized in ● Compared with recently published papers describing the pharmacokinetic profiles of phentolamine in healthy volunteers, the pharmacokinetic parameters of phentolamine obtained in the study were generally similar [15].
Conclusions
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