Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 85–89
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Simultaneous determination of 7-O-succinyl macrolactin A and its metabolite macrolactin A in rat plasma using liquid chromatography coupled to tandem mass spectrometry Keumhan Noh a , Dong Hee Kim b , Beom Soo Shin c , Hwi-yeol Yun d , Eunyoung Kim e,∗∗ , Wonku Kang e,∗ a
College of Pharmacy, Yeungnam University, Kyoungbuk 712-749, South Korea Research and Development Center, Daewoo Pharm. Co. Ltd., Busan 604-836, South Korea c College of Pharmacy, Catholic University of Daegu, Kyoungbuk 712-702, South Korea d College of Pharmacy, Chungnam National University, Daejeon 305-764, South Korea e College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea b
a r t i c l e
i n f o
Article history: Received 11 February 2014 Received in revised form 7 May 2014 Accepted 9 May 2014 Available online 17 May 2014 Keywords: 7-O-Succinyl macrolactin A Macrolactin A LC–MS/MS Rat plasma Stability
a b s t r a c t 7-O-Succinyl macrolactin A (SMA) and its major metabolite macrolactin A (MA) are generated from Bacillus polyfermenticus KJS-2. Both substances show inhibitory effects on angiogenesis and cancer cell invasion. SMA in rat plasma is known to be relatively stable at room temperature, but MA was not detected due to its instability. Therefore, a stabilizer is required to accurately measure the substance in biological rat samples. In this study, NaF and eserine were examined to determine whether they could stabilize MA to allow for accurate measurement in rat plasma. We also developed a rapid and simple chromatographic method using tandem mass spectrometry (MS/MS) for the simultaneous determination of these compounds in rat plasma. After simple protein precipitation with acetonitrile including methaqualone (internal standard), the analytes were chromatographed on a Hilic column with a mobile phase of 10 mM formic acid aqueous solution, methanol, and acetonitrile (15:15:70, v/v). The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This analytical method was successfully applied to monitor plasma concentrations of both compounds over time following intravenous administration of a salt form of SMA in rats. © 2014 Elsevier B.V. All rights reserved.
1. Introduction 7-O-Succinyl macrolactin A (SMA) and its major metabolite macrolactin A (MA) are generated from Bacillus polyfermenticus KJS-2 [1]. Both substances have antibacterial activities against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus [1,2], and show inhibitory effects on angiogenesis and cancer cell invasion [3]. During the drug development process, pharmacokinetic studies are required to evaluate drug clearance and metabolism in the body. However, an appropriate analytical method is required prior to performing pharmacokinetic studies. At this time, SMA has been measured in rat plasma and urine using high-performance liquid chromatography (HPLC) with UV detection by Kim et al. [4].
∗ Corresponding author. Tel.: +82 28205601; fax: +82 28167338. ∗∗ Corresponding author. E-mail addresses: [email protected] (E. Kim), [email protected] (W. Kang). http://dx.doi.org/10.1016/j.jpba.2014.05.009 0731-7085/© 2014 Elsevier B.V. All rights reserved.
They reported that SMA in rat plasma is stable for only 2 h at room temperature, and that MA was not detected due to its instability. As shown in Fig. 1, the ester in the ring structure of MA seems to be easily hydrolyzed by esterases in biological fluids. Therefore, a stabilizer is required to accurately measure the substance in biological rat samples. In general, rat plasma contains four esterases: acetylcholinesterase (AChE), arylesterase (ArE), butyrylcholinesterase (BChE), and carboxylesterase (CES) [5]. Eserine can selectively inhibit AChE and BChE, sodium fluoride (NaF) inhibits AchE, BChE, and CES, and metal chlorides inhibit ArE [6,7]. In this study, Naf and eserine were examined to determine whether they could stabilize both SMA and MA to allow for accurate measurement in rat plasma. We also developed a rapid and simple chromatographic method using tandem mass spectrometry (MS/MS) for the simultaneous determination of these compounds in rat plasma. This analytical method was successfully applied to monitor plasma concentrations of both compounds over time following intravenous administration of a salt form of SMA in rats.
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K. Noh et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 85–89
Fig. 1. Chemical structures and chromatograms of 7-O-succinyl macrolactin A (SMA), macrolactin A (MA) and methaqualone. Top, double-blank plasma; middle, plasma spiked with 0.1 g/ml SMA and MA, and 1 ng/ml methaqualone (IS); bottom, a plasma sample equivalent to 1.13 g/ml for SMA and 0.09 g/ml for MA, respectively, in a sample obtained from a rat at 60 min after an intravenous injection of 90 mg/kg a salt of SMA.
2. Materials and methods
positive-ion mode. The major peaks in the MS/MS scan were used to quantify the three compounds.
2.1. Reagents and materials SMA and MA were kindly donated by Daewoo Pharmaceutical Ind. Co. Ltd. (Busan, Korea). Methaqualone (internal standard, IS), NaF, and eserine were purchased from Sigma (Seoul, Korea), and acetonitrile was obtained from Burdick & Jackson (Muskegon, MI, USA). All other chemicals and solvents were of the highest analytical grade available. 2.2. Preparation of standards and quality controls SMA, MA, and the IS were dissolved in methanol to a concentration of 1.0 mg/ml. SMA and MA solutions were then serially diluted with methanol, and 5 l of a mixture of both diluted solutions was added to 45 l of drug-free plasma to obtain final concentrations of 0.02, 0.05, 0.1, 0.5, 1, and 10 g/ml for SMA, and 0.02, 0.05, 0.1, 0.5, 1, and 5 g/ml for MA. Using linear regression, six calibration graphs were derived from the ratio between the area under the peak of each compound and the IS. Quality control samples were prepared in 45 l of blank rat plasma by adding 5 l of serially diluted solutions of each substance to determine the lower limit of quantification (0.02 g/ml for both), as well as low (0.06 g/ml for both), intermediate (0.45 g/ml for SMA and 0.22 g/ml for MA), and high concentrations (9 g/ml for SMA and 4.5 g/ml for MA). These samples were used to evaluate the intra- and inter-day precision and accuracy of the assay.
2.4. Analytical system Plasma concentrations of SMA and MA were quantified using an API 4000 LC/MS/MS system (AB SCIEX, Framingham, MA, USA) equipped with an electrospray ionization interface in positive-ion mode. The compounds were separated on a reversed-phase column (ZIC® Hilic, 50 × 2.1 mm internal diameter, 5-m particle size; Merck, Darmstadt, Germany) in the mobile phase (10 mM formic acid aqueous solution, methanol, and acetonitrile, 15:15:70, v/v/v). The column was heated to 40 ◦ C, and the mobile phase was eluted at 0.3 ml/min using an HP 1260 series pump (Agilent, Wilmington, DE, USA). The turbo ion spray interface was operated in positive-ion mode at 5500 V and 450 ◦ C. SMA and MA were mainly identified as sodium adduct ions [M+Na]+ at m/z 525.4 and 425.3, respectively, and the IS produced primarily protonated molecules [M+H]+ at m/z 251.1. The product ions were scanned in Q3 after collision with nitrogen in Q2 at m/z 406.7, 258.7, and 132.1 for SMA, MA, and the IS, respectively. Quantification was performed by selective reaction monitoring of the sodium adducts or protonated precursor ions and the related product ions using the ratio of the area under the peak for each solution. The analytical data were processed using Analyst software (ver. 1.5.2, Applied Biosystems, Foster City, CA, USA).
2.3. Characterization of product ions using MS/MS
2.5. Sample preparation
Briefly, 100 ng/ml of SMA and MA, and 10 ng/ml of the IS were individually infused into the mass spectrometer at a flow rate of 10 l/min to characterize the product ions of each substance. The precursor ions and fragmentation patterns were monitored using
The IS (250 l; 1 ng/ml in methanol) was added to 50 l of rat and spiked plasma, vortex-mixed for 10 s, and centrifuged (13,200 rpm, 10 min). A 3-l aliquot of the supernatant was then injected into the column [8,9].
K. Noh et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 85–89
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Table 1 Precision and accuracy of the intra- and inter day assay (n = 5). Concentration (g/ml)
SMA
Concentration (g/ml)
MA
Intra-day
Inter-day
0.02
101.3 ± 9.1a (9.0)b
106.6 ± 5.9 (5.6)
0.02
101.9 ± 5.8 (5.7)
92.5 ± 7.2 (7.8)
0.06
100.1 ± 2.3 (2.3)
95.8 ± 3.6 (3.8)
0.06
106.8 ± 7.2 (6.7)
107.7 ± 3.4 (3.2)
0.45
96.9 ± 3.0 (3.1)
97.6 ± 3.1 (3.2)
0.22
106.9 ± 4.6 (4.3)
103.6 ± 3.3 (3.2)
101.4 ± 8.5 (8.4)
100.4 ± 2.2 (2.2)
96.1 ± 6.2 (6.5)
98.2 ± 2.6 (2.6)
9 a b
Intra-day
4.5
Inter-day
Accuracy (mean% ± S.D.). RSD, relative standard deviation (%).
2.6. Validation procedure The validation parameters were selectivity, extraction recovery, precision, and accuracy. Blank plasma samples obtained from six rats were screened to determine specificity. The intra- and interday assay precision and accuracy were estimated using a calibration curve to predict the concentration of quality controls. Acceptable criteria were within 15% of precision and accuracy, except the lower limit of quantification, which was within 20%. Recovery was determined by comparing the mean peak areas of quality controls spiked before protein precipitation to those spiked after pretreatment. The matrix effect was assessed based on the percentile of the mean peak areas of quality controls spiked after pretreatment of stock solutions. The dilution effect for SMA for samples out of range of the standard curve was evaluated after 20- and 100-fold dilutions with blank rat plasma. 2.7. Stabilizing effect of esterase inhibitors NaF or eserine was added to plasma as an esterase inhibitor to determine whether they could prevent decomposition of SMA and MA in rat plasma. Fresh rat plasma (45 l) drawn from SpragueDawley rats was mixed with 5 l of distilled water (control), NaF (24 mM final concentration), or eserine (0.1 mM final concentration), and after 5 min at 37 ◦ C for the preincubation, 5 l of 10 g/ml SMA or MA solutions was added. The stabilizing effects of both NaF and eserine were serially monitored for 2 h, and the incubation was stopped by the addition of 250 l methanol (including the IS). 2.8. Stability Stability of stock solutions and plasma samples was examined under different conditions. The stock solution was checked for short-term stability after 4 h of storage at room temperature and for long-term stability after 4 weeks at 4 ◦ C. For the stability study in plasma, control samples were made at 0.1 and 1 g/ml concentrations, and NaF was added to plasma samples to prevent enzymatic degradation of the analytes. Short- and long-term stabilities were assessed after 2 h of storage at room temperature and after 2 weeks of storage at −70 ◦ C, respectively. The stability of SMA and MA in plasma samples was also tested after three freeze–thaw cycles (−70 ◦ C to room temperature). The stability of the three compounds in extracts was also examined after 24 h of storage at 4 ◦ C. The requirement for a stable analyte was that the difference between the mean concentrations of tested samples after being placed under various conditions was approximately ±15%. 2.9. Application Five Sprague-Dawley rats were administered a single intravenous injection of 90 mg/kg of an SMA salt. Blood samples (0.2 ml) were serially taken for up to 2 h after drug administration and collected into heparinized centrifuge tubes containing NaF. After
centrifugation at 13,200 rpm for 10 min, plasma samples were immediately analyzed as described above. 3. Results and discussion 3.1. Quantification of compounds and method validation SMA and MA predominantly produced sodium adduct molecules at m/z 525.4 and 425.3, respectively, while the IS generated protonated ions at m/z 251.1. After collision with N2 in Q2, the corresponding product ions were scanned at m/z 406.7 for SMA, 258.7 for MA, and 132.1 for the IS in Q3. Fig. 1 presents a typical chromatogram for blank plasma (top), plasma spiked with 100 ng/ml of SMA and MA plus 1 ng/ml of the IS (middle), and a rat plasma sample (bottom). Although the analytes were eluted relatively earlier at 1.5 min because a hydrophilic interactive LC column was used, the chromatographic run time was 10 min to rule out carryover effects. The calibration curves provided a reliable response for the two compounds. The ratio of the peak area of SMA and MA relative to that of the IS was correlated with the corresponding plasma concentration, and good linearity was observed (r2 > 0.997). The detection limits for SMA and MA were 4 and 10 ng/ml at a signal-to-noise (S/N) ratio of 3, respectively. The relative standard deviations of the intraday assay precision were less than 9.0% and 6.7% for SMA and MA, respectively. The intraday assay accuracy was 96.9–101.4% for SMA and 96.1–106.9% for MA. The relative standard deviations of the inter-day precision were less than 5.6% for SMA and 7.8% for MA. The inter-day accuracy was 95.8–106.6% for SMA and 92.5–107.7% for MA (Table 1). The mean recoveries of SMA and MA ranged from 99% to 102% and 86%–93%, respectively, and the mean matrix effect was 97–102% for SMA and 66–77% for MA (Table 2). No dilution effect for SMA was found at 20- and 100-fold dilutions with blank rat plasma. 3.2. Effect of esterase inhibitors on SMA and MA stability Fig. 2A and B shows the stability of SMA and MA in plasma in the presence and absence of esterase inhibitors, NaF (24 mM), or serine (0.1 mM). SMA was stable up to 2 h in the presence or absence of esterase inhibitors, which was consistent with previous results published by Kim et al. [4]. However, more than 50% of MA was rapidly degraded within 0.5 h in the absence of NaF and eserine, while over 80% of MA remained for 1 h in plasma in the presence of esterase inhibitors. NaF showed better protective effects (87%) against esterases than eserine (82%), perhaps because NaF blocked carboxylesterase, a primary esterase in rat plasma, while eserine did not. NaF and eserine have been previously examined at 1.57–240 mM [7,10,11] and 0.1 mM [12], respectively. Three times higher concentration (72 mM) of NaF was also tested in this study, but it did not improve the stability of the test items. Carboxyesterase activity in rat plasma is known to be greater than that in dog and human plasma, and MA is relatively more stable in
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Table 2 Matrix effect and recovery for SMA and MA in rat plasma (mean% ± S.D., n = 5). Concentration (g/ml)
SMA
Concentration (g/ml)
Matrix effect (%) 0.02 0.06 0.45 9
102.0 97.0 97.2 97.4
± ± ± ±
Recovery (%)
4.0 7.6 3.9 1.6
102.0 99.0 99.3 101.3
± ± ± ±
12.7 6.5 2.7 2.1
MA Matrix effect (%)
0.02 0.06 0.22 4.5
77.0 68.6 67.9 65.8
± ± ± ±
3.7 2.8 4.7 2.9
Recovery (%) 87.0 86.1 93.4 90.1
± ± ± ±
5.9 3.8 6.6 1.7
Fig. 2. Stability of 1 g/ml SMA (A) and MA (B) in rat plasma in the presence and absence (control) of NaF (24 mM) or eserine (0.1 mM), and time courses of plasma SMA (C) and MA (D) concentrations in rats after a single intravenous injection of 90 mg/kg a salt of SMA (n = 5). The results are expressed as means and standard deviations. Student t-test was used to evaluate differences compare to control.
dog plasma compared to rat plasma (data not shown). The species differences in enzyme activity should be taken into account to predict the biotransformation of compounds, including ester moieties [10]. In addition, an ester out of the ring may be more stable than esters in the ring, and perhaps conformational changes in the side chains could prevent enzymatic degradation of the latter ester. We explored whether other metabolites of SMA (e.g., ring-opened structures of MA and/or SMA) were present using MS. However, the simple ring-opened molecules of SMA and MA were not detected, suggesting that they may be further biotransformed followed by ring-opening.
3.3. Stability SMA and MA stock solutions were stable at room temperature for 4 h and at 4 ◦ C for 4 weeks. MA in rat plasma required NaF under all conditions to ensure stability during pretreatment and storage (Table 3). SMA and MA in plasma were stable for up to 2 and 1 h at room temperature, respectively, and remained intact for up to 2 weeks for SMA and 1 week for MA at −70 ◦ C. No degradation was observed after three cycles of freezing and thawing. The stability of the compounds in extracts was confirmed after 24 h of storage at 4 ◦ C.
Table 3 Stability of SMA and MA after storage under indicated condition (mean% ± S.D., n = 3). Storage condition
SMA 0.1 g/ml
Standard solutions Room temperature (4 h) Refrigerator (4 weeks) Plasma samples Room temperaturea Refrigerator in extracts (24 h) 3 cycles of freezing–thawing −70 ◦ C (2 weeks for SMA; 1week for MA) a
4 h for SMA and 50 min for MA.
88.9 ± 4.2 95.3 ± 2.4 97.3 ± 7.4 105.2 ± 4.0 87.4 ± 1.9 106.0 ± 2.6
MA 1 g/ml
0.1 g/ml
100.2 ± 5.5 103.2 ± 1.1
92.3 ± 6.8 102.9 ± 2.6
102.3 108.1 92.9 108.5
± ± ± ±
10.7 1.8 2.0 3.4
87.8 ± 2.5 99.1 ± 3.2 106.8 ± 8.9 111.9 ± 2.0
1 g/ml 94.7 ± 3.1 89.1 ± 0.3 86.8 99.5 110.5 110.1
± ± ± ±
3.2 0.7 3.6 1.0
K. Noh et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 85–89
3.4. Application of the method The validated method described above was used to evaluate the pharmacokinetics of SMA and its metabolite MA in rats. Fig. 2C and D shows the mean plasma concentrations of SMA and MA after a single intravenous injection of 90 mg/kg of an SMA salt in five rats, respectively. The volume of distribution of SMA was 0.2 L/kg, and the half-life was about 15 min. MA concentration peaked (0.7 g/ml) approximately 20 min following intravenous administration of the SMA prodrug, and decayed with a 17 min half-life. 4. Conclusions SMA is stable in rat plasma, but its major active metabolite MA is unstable. Therefore, an esterase inhibitor such as NaF is required in plasma to ensure MA stability during pretreatment and storage. A sensitive method for the simultaneous determination of SMA and MA in rat plasma was developed using LC/MS/MS. This method is suitable for pharmacokinetic studies of both SMA and MA in rats. Acknowledgements
[2]
[3] [4]
[5]
[6]
[7] [8]
[9]
[10]
This research was supported by grants from Daewoo Pharmaceutical Co. Ltd. and National Research Foundation of Korea (2010-0003486).
[11]
References
[12]
[1] D.H. Kim, H.K. Kim, K.M. Kim, C.K. Kim, M.H. Jeong, C.Y. Ko, K.H. Moon, J.S. Kang, Antibacterial activities of macrolactin A and 7-O-succinyl macrolactin A
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from Bacillus polyfermenticus KJS-2 against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus, Arch. Pharm. Res. 34 (2011) 147–152. M. Romero-Tabarez, R. Jansen, M. Sylla, H. Lünsdorf, S. Häussler, D.A. Santosa, K.N. Timmis, G. Molinari, 7-O-Malonyl macrolactin A, a new macrolactin antibiotic from Bacillus subtilis active against methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and a small-colony variant of Burkholderia cepacia, Antimicrob. Agents Chemother. 50 (2006) 1701– 1709. S.U. Chung, S.W. Hwang, Y.H. Ji, J.S. Kang, K.R. Kang, Y.R. Kang, D.H. Kim, J.A. Kim, Patent WO2012008674 A1 (2012). J.M. Kim, J.W. Jung, D.H. Kim, J.S. Kang, C.G. Kim, H.E. Kang, A simple and sensitive HPLC-UV determination of 7-O-succinyl macrolactin A in rat plasma and urine and its application to a pharmacokinetic study, Biomed. Chromatogr. 27 (2013) 273–279. W. Li, J. Zhang, F.L. Tse, Strategies in quantitative LC–MS/MS analysis of unstable small molecules in biological matrices, Biomed. Chromatogr. 25 (2011) 258–277. J.G. Slatter, P. Su, J.P. Sams, L.J. Schaaf, L.C. Wienkers, Bioactivation of the anticancer agent CPT-11 to SN-38 by human hepatic microsomal carboxylesterases and the in vitro assessment of potential drug interactions, Drug Metab. Dispos. 25 (1997) 1157–1164. R.M. Krupka, Fluoride inhibition of acetylcholinesterase, Mol. Pharmacol. 2 (1996) 558–569. K. Noh, J.H. Park, M. Kim, M. Jung, H. Ha, K.I. Kwon, H.J. Lee, W. Kang, Quantitative determination of daumone in rat plasma by liquid chromatography–mass spectrometry, J. Pharm. Biomed. Anal. 56 (2011) 114–117. W. Kang, E.Y. Kim, Simultaneous determination of aceclofenac and its three metabolites in plasma using liquid chromatography-tandem mass spectrometry, J. Pharm. Biomed. Anal. 46 (2008) 587–591. ˜ S.W. Toennes, G.F. Kauert, Studies on in vitro degradation of A.S. Fandino, anhydroecgonine methyl ester (methylecgonidine) in human plasma, J. Anal. Toxicol. 26 (2002) 567–570. R.W. Dulac, T.J. Yang, Differential sodium fluoride sensitivity of a-naphthyl acetate esterase in human, bovine, canine and murine monocytes and lymphocytes, Exp. Hematol. 19 (1991) 59–62. E. Wahlin-Boll, B. Brantmark, A. Hanson, A. Melander, C. Nilsson, High-pressure liquid chromatographic determination of acetylsalicylic acid, salicylic acid, diflunisal, indomethacin, indoprofen and indobufen, Eur. J. Clin. Pharmacol. 20 (1981) 375–378.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Simultaneous determination of 7-O-succinyl macrolactin A and its metabolite macrolactin A in rat plasma using liquid chromatography coupled to tandem mass spectrometry Keumhan Noh a , Dong Hee Kim b , Beom Soo Shin c , Hwi-yeol Yun d , Eunyoung Kim e,∗∗ , Wonku Kang e,∗ a
College of Pharmacy, Yeungnam University, Kyoungbuk 712-749, South Korea Research and Development Center, Daewoo Pharm. Co. Ltd., Busan 604-836, South Korea c College of Pharmacy, Catholic University of Daegu, Kyoungbuk 712-702, South Korea d College of Pharmacy, Chungnam National University, Daejeon 305-764, South Korea e College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea b
a r t i c l e
i n f o
Article history: Received 11 February 2014 Received in revised form 7 May 2014 Accepted 9 May 2014 Available online 17 May 2014 Keywords: 7-O-Succinyl macrolactin A Macrolactin A LC–MS/MS Rat plasma Stability
a b s t r a c t 7-O-Succinyl macrolactin A (SMA) and its major metabolite macrolactin A (MA) are generated from Bacillus polyfermenticus KJS-2. Both substances show inhibitory effects on angiogenesis and cancer cell invasion. SMA in rat plasma is known to be relatively stable at room temperature, but MA was not detected due to its instability. Therefore, a stabilizer is required to accurately measure the substance in biological rat samples. In this study, NaF and eserine were examined to determine whether they could stabilize MA to allow for accurate measurement in rat plasma. We also developed a rapid and simple chromatographic method using tandem mass spectrometry (MS/MS) for the simultaneous determination of these compounds in rat plasma. After simple protein precipitation with acetonitrile including methaqualone (internal standard), the analytes were chromatographed on a Hilic column with a mobile phase of 10 mM formic acid aqueous solution, methanol, and acetonitrile (15:15:70, v/v). The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This analytical method was successfully applied to monitor plasma concentrations of both compounds over time following intravenous administration of a salt form of SMA in rats. © 2014 Elsevier B.V. All rights reserved.
1. Introduction 7-O-Succinyl macrolactin A (SMA) and its major metabolite macrolactin A (MA) are generated from Bacillus polyfermenticus KJS-2 [1]. Both substances have antibacterial activities against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus [1,2], and show inhibitory effects on angiogenesis and cancer cell invasion [3]. During the drug development process, pharmacokinetic studies are required to evaluate drug clearance and metabolism in the body. However, an appropriate analytical method is required prior to performing pharmacokinetic studies. At this time, SMA has been measured in rat plasma and urine using high-performance liquid chromatography (HPLC) with UV detection by Kim et al. [4].
∗ Corresponding author. Tel.: +82 28205601; fax: +82 28167338. ∗∗ Corresponding author. E-mail addresses: [email protected] (E. Kim), [email protected] (W. Kang). http://dx.doi.org/10.1016/j.jpba.2014.05.009 0731-7085/© 2014 Elsevier B.V. All rights reserved.
They reported that SMA in rat plasma is stable for only 2 h at room temperature, and that MA was not detected due to its instability. As shown in Fig. 1, the ester in the ring structure of MA seems to be easily hydrolyzed by esterases in biological fluids. Therefore, a stabilizer is required to accurately measure the substance in biological rat samples. In general, rat plasma contains four esterases: acetylcholinesterase (AChE), arylesterase (ArE), butyrylcholinesterase (BChE), and carboxylesterase (CES) [5]. Eserine can selectively inhibit AChE and BChE, sodium fluoride (NaF) inhibits AchE, BChE, and CES, and metal chlorides inhibit ArE [6,7]. In this study, Naf and eserine were examined to determine whether they could stabilize both SMA and MA to allow for accurate measurement in rat plasma. We also developed a rapid and simple chromatographic method using tandem mass spectrometry (MS/MS) for the simultaneous determination of these compounds in rat plasma. This analytical method was successfully applied to monitor plasma concentrations of both compounds over time following intravenous administration of a salt form of SMA in rats.
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K. Noh et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 85–89
Fig. 1. Chemical structures and chromatograms of 7-O-succinyl macrolactin A (SMA), macrolactin A (MA) and methaqualone. Top, double-blank plasma; middle, plasma spiked with 0.1 g/ml SMA and MA, and 1 ng/ml methaqualone (IS); bottom, a plasma sample equivalent to 1.13 g/ml for SMA and 0.09 g/ml for MA, respectively, in a sample obtained from a rat at 60 min after an intravenous injection of 90 mg/kg a salt of SMA.
2. Materials and methods
positive-ion mode. The major peaks in the MS/MS scan were used to quantify the three compounds.
2.1. Reagents and materials SMA and MA were kindly donated by Daewoo Pharmaceutical Ind. Co. Ltd. (Busan, Korea). Methaqualone (internal standard, IS), NaF, and eserine were purchased from Sigma (Seoul, Korea), and acetonitrile was obtained from Burdick & Jackson (Muskegon, MI, USA). All other chemicals and solvents were of the highest analytical grade available. 2.2. Preparation of standards and quality controls SMA, MA, and the IS were dissolved in methanol to a concentration of 1.0 mg/ml. SMA and MA solutions were then serially diluted with methanol, and 5 l of a mixture of both diluted solutions was added to 45 l of drug-free plasma to obtain final concentrations of 0.02, 0.05, 0.1, 0.5, 1, and 10 g/ml for SMA, and 0.02, 0.05, 0.1, 0.5, 1, and 5 g/ml for MA. Using linear regression, six calibration graphs were derived from the ratio between the area under the peak of each compound and the IS. Quality control samples were prepared in 45 l of blank rat plasma by adding 5 l of serially diluted solutions of each substance to determine the lower limit of quantification (0.02 g/ml for both), as well as low (0.06 g/ml for both), intermediate (0.45 g/ml for SMA and 0.22 g/ml for MA), and high concentrations (9 g/ml for SMA and 4.5 g/ml for MA). These samples were used to evaluate the intra- and inter-day precision and accuracy of the assay.
2.4. Analytical system Plasma concentrations of SMA and MA were quantified using an API 4000 LC/MS/MS system (AB SCIEX, Framingham, MA, USA) equipped with an electrospray ionization interface in positive-ion mode. The compounds were separated on a reversed-phase column (ZIC® Hilic, 50 × 2.1 mm internal diameter, 5-m particle size; Merck, Darmstadt, Germany) in the mobile phase (10 mM formic acid aqueous solution, methanol, and acetonitrile, 15:15:70, v/v/v). The column was heated to 40 ◦ C, and the mobile phase was eluted at 0.3 ml/min using an HP 1260 series pump (Agilent, Wilmington, DE, USA). The turbo ion spray interface was operated in positive-ion mode at 5500 V and 450 ◦ C. SMA and MA were mainly identified as sodium adduct ions [M+Na]+ at m/z 525.4 and 425.3, respectively, and the IS produced primarily protonated molecules [M+H]+ at m/z 251.1. The product ions were scanned in Q3 after collision with nitrogen in Q2 at m/z 406.7, 258.7, and 132.1 for SMA, MA, and the IS, respectively. Quantification was performed by selective reaction monitoring of the sodium adducts or protonated precursor ions and the related product ions using the ratio of the area under the peak for each solution. The analytical data were processed using Analyst software (ver. 1.5.2, Applied Biosystems, Foster City, CA, USA).
2.3. Characterization of product ions using MS/MS
2.5. Sample preparation
Briefly, 100 ng/ml of SMA and MA, and 10 ng/ml of the IS were individually infused into the mass spectrometer at a flow rate of 10 l/min to characterize the product ions of each substance. The precursor ions and fragmentation patterns were monitored using
The IS (250 l; 1 ng/ml in methanol) was added to 50 l of rat and spiked plasma, vortex-mixed for 10 s, and centrifuged (13,200 rpm, 10 min). A 3-l aliquot of the supernatant was then injected into the column [8,9].
K. Noh et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 85–89
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Table 1 Precision and accuracy of the intra- and inter day assay (n = 5). Concentration (g/ml)
SMA
Concentration (g/ml)
MA
Intra-day
Inter-day
0.02
101.3 ± 9.1a (9.0)b
106.6 ± 5.9 (5.6)
0.02
101.9 ± 5.8 (5.7)
92.5 ± 7.2 (7.8)
0.06
100.1 ± 2.3 (2.3)
95.8 ± 3.6 (3.8)
0.06
106.8 ± 7.2 (6.7)
107.7 ± 3.4 (3.2)
0.45
96.9 ± 3.0 (3.1)
97.6 ± 3.1 (3.2)
0.22
106.9 ± 4.6 (4.3)
103.6 ± 3.3 (3.2)
101.4 ± 8.5 (8.4)
100.4 ± 2.2 (2.2)
96.1 ± 6.2 (6.5)
98.2 ± 2.6 (2.6)
9 a b
Intra-day
4.5
Inter-day
Accuracy (mean% ± S.D.). RSD, relative standard deviation (%).
2.6. Validation procedure The validation parameters were selectivity, extraction recovery, precision, and accuracy. Blank plasma samples obtained from six rats were screened to determine specificity. The intra- and interday assay precision and accuracy were estimated using a calibration curve to predict the concentration of quality controls. Acceptable criteria were within 15% of precision and accuracy, except the lower limit of quantification, which was within 20%. Recovery was determined by comparing the mean peak areas of quality controls spiked before protein precipitation to those spiked after pretreatment. The matrix effect was assessed based on the percentile of the mean peak areas of quality controls spiked after pretreatment of stock solutions. The dilution effect for SMA for samples out of range of the standard curve was evaluated after 20- and 100-fold dilutions with blank rat plasma. 2.7. Stabilizing effect of esterase inhibitors NaF or eserine was added to plasma as an esterase inhibitor to determine whether they could prevent decomposition of SMA and MA in rat plasma. Fresh rat plasma (45 l) drawn from SpragueDawley rats was mixed with 5 l of distilled water (control), NaF (24 mM final concentration), or eserine (0.1 mM final concentration), and after 5 min at 37 ◦ C for the preincubation, 5 l of 10 g/ml SMA or MA solutions was added. The stabilizing effects of both NaF and eserine were serially monitored for 2 h, and the incubation was stopped by the addition of 250 l methanol (including the IS). 2.8. Stability Stability of stock solutions and plasma samples was examined under different conditions. The stock solution was checked for short-term stability after 4 h of storage at room temperature and for long-term stability after 4 weeks at 4 ◦ C. For the stability study in plasma, control samples were made at 0.1 and 1 g/ml concentrations, and NaF was added to plasma samples to prevent enzymatic degradation of the analytes. Short- and long-term stabilities were assessed after 2 h of storage at room temperature and after 2 weeks of storage at −70 ◦ C, respectively. The stability of SMA and MA in plasma samples was also tested after three freeze–thaw cycles (−70 ◦ C to room temperature). The stability of the three compounds in extracts was also examined after 24 h of storage at 4 ◦ C. The requirement for a stable analyte was that the difference between the mean concentrations of tested samples after being placed under various conditions was approximately ±15%. 2.9. Application Five Sprague-Dawley rats were administered a single intravenous injection of 90 mg/kg of an SMA salt. Blood samples (0.2 ml) were serially taken for up to 2 h after drug administration and collected into heparinized centrifuge tubes containing NaF. After
centrifugation at 13,200 rpm for 10 min, plasma samples were immediately analyzed as described above. 3. Results and discussion 3.1. Quantification of compounds and method validation SMA and MA predominantly produced sodium adduct molecules at m/z 525.4 and 425.3, respectively, while the IS generated protonated ions at m/z 251.1. After collision with N2 in Q2, the corresponding product ions were scanned at m/z 406.7 for SMA, 258.7 for MA, and 132.1 for the IS in Q3. Fig. 1 presents a typical chromatogram for blank plasma (top), plasma spiked with 100 ng/ml of SMA and MA plus 1 ng/ml of the IS (middle), and a rat plasma sample (bottom). Although the analytes were eluted relatively earlier at 1.5 min because a hydrophilic interactive LC column was used, the chromatographic run time was 10 min to rule out carryover effects. The calibration curves provided a reliable response for the two compounds. The ratio of the peak area of SMA and MA relative to that of the IS was correlated with the corresponding plasma concentration, and good linearity was observed (r2 > 0.997). The detection limits for SMA and MA were 4 and 10 ng/ml at a signal-to-noise (S/N) ratio of 3, respectively. The relative standard deviations of the intraday assay precision were less than 9.0% and 6.7% for SMA and MA, respectively. The intraday assay accuracy was 96.9–101.4% for SMA and 96.1–106.9% for MA. The relative standard deviations of the inter-day precision were less than 5.6% for SMA and 7.8% for MA. The inter-day accuracy was 95.8–106.6% for SMA and 92.5–107.7% for MA (Table 1). The mean recoveries of SMA and MA ranged from 99% to 102% and 86%–93%, respectively, and the mean matrix effect was 97–102% for SMA and 66–77% for MA (Table 2). No dilution effect for SMA was found at 20- and 100-fold dilutions with blank rat plasma. 3.2. Effect of esterase inhibitors on SMA and MA stability Fig. 2A and B shows the stability of SMA and MA in plasma in the presence and absence of esterase inhibitors, NaF (24 mM), or serine (0.1 mM). SMA was stable up to 2 h in the presence or absence of esterase inhibitors, which was consistent with previous results published by Kim et al. [4]. However, more than 50% of MA was rapidly degraded within 0.5 h in the absence of NaF and eserine, while over 80% of MA remained for 1 h in plasma in the presence of esterase inhibitors. NaF showed better protective effects (87%) against esterases than eserine (82%), perhaps because NaF blocked carboxylesterase, a primary esterase in rat plasma, while eserine did not. NaF and eserine have been previously examined at 1.57–240 mM [7,10,11] and 0.1 mM [12], respectively. Three times higher concentration (72 mM) of NaF was also tested in this study, but it did not improve the stability of the test items. Carboxyesterase activity in rat plasma is known to be greater than that in dog and human plasma, and MA is relatively more stable in
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K. Noh et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 85–89
Table 2 Matrix effect and recovery for SMA and MA in rat plasma (mean% ± S.D., n = 5). Concentration (g/ml)
SMA
Concentration (g/ml)
Matrix effect (%) 0.02 0.06 0.45 9
102.0 97.0 97.2 97.4
± ± ± ±
Recovery (%)
4.0 7.6 3.9 1.6
102.0 99.0 99.3 101.3
± ± ± ±
12.7 6.5 2.7 2.1
MA Matrix effect (%)
0.02 0.06 0.22 4.5
77.0 68.6 67.9 65.8
± ± ± ±
3.7 2.8 4.7 2.9
Recovery (%) 87.0 86.1 93.4 90.1
± ± ± ±
5.9 3.8 6.6 1.7
Fig. 2. Stability of 1 g/ml SMA (A) and MA (B) in rat plasma in the presence and absence (control) of NaF (24 mM) or eserine (0.1 mM), and time courses of plasma SMA (C) and MA (D) concentrations in rats after a single intravenous injection of 90 mg/kg a salt of SMA (n = 5). The results are expressed as means and standard deviations. Student t-test was used to evaluate differences compare to control.
dog plasma compared to rat plasma (data not shown). The species differences in enzyme activity should be taken into account to predict the biotransformation of compounds, including ester moieties [10]. In addition, an ester out of the ring may be more stable than esters in the ring, and perhaps conformational changes in the side chains could prevent enzymatic degradation of the latter ester. We explored whether other metabolites of SMA (e.g., ring-opened structures of MA and/or SMA) were present using MS. However, the simple ring-opened molecules of SMA and MA were not detected, suggesting that they may be further biotransformed followed by ring-opening.
3.3. Stability SMA and MA stock solutions were stable at room temperature for 4 h and at 4 ◦ C for 4 weeks. MA in rat plasma required NaF under all conditions to ensure stability during pretreatment and storage (Table 3). SMA and MA in plasma were stable for up to 2 and 1 h at room temperature, respectively, and remained intact for up to 2 weeks for SMA and 1 week for MA at −70 ◦ C. No degradation was observed after three cycles of freezing and thawing. The stability of the compounds in extracts was confirmed after 24 h of storage at 4 ◦ C.
Table 3 Stability of SMA and MA after storage under indicated condition (mean% ± S.D., n = 3). Storage condition
SMA 0.1 g/ml
Standard solutions Room temperature (4 h) Refrigerator (4 weeks) Plasma samples Room temperaturea Refrigerator in extracts (24 h) 3 cycles of freezing–thawing −70 ◦ C (2 weeks for SMA; 1week for MA) a
4 h for SMA and 50 min for MA.
88.9 ± 4.2 95.3 ± 2.4 97.3 ± 7.4 105.2 ± 4.0 87.4 ± 1.9 106.0 ± 2.6
MA 1 g/ml
0.1 g/ml
100.2 ± 5.5 103.2 ± 1.1
92.3 ± 6.8 102.9 ± 2.6
102.3 108.1 92.9 108.5
± ± ± ±
10.7 1.8 2.0 3.4
87.8 ± 2.5 99.1 ± 3.2 106.8 ± 8.9 111.9 ± 2.0
1 g/ml 94.7 ± 3.1 89.1 ± 0.3 86.8 99.5 110.5 110.1
± ± ± ±
3.2 0.7 3.6 1.0
K. Noh et al. / Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 85–89
3.4. Application of the method The validated method described above was used to evaluate the pharmacokinetics of SMA and its metabolite MA in rats. Fig. 2C and D shows the mean plasma concentrations of SMA and MA after a single intravenous injection of 90 mg/kg of an SMA salt in five rats, respectively. The volume of distribution of SMA was 0.2 L/kg, and the half-life was about 15 min. MA concentration peaked (0.7 g/ml) approximately 20 min following intravenous administration of the SMA prodrug, and decayed with a 17 min half-life. 4. Conclusions SMA is stable in rat plasma, but its major active metabolite MA is unstable. Therefore, an esterase inhibitor such as NaF is required in plasma to ensure MA stability during pretreatment and storage. A sensitive method for the simultaneous determination of SMA and MA in rat plasma was developed using LC/MS/MS. This method is suitable for pharmacokinetic studies of both SMA and MA in rats. Acknowledgements
[2]
[3] [4]
[5]
[6]
[7] [8]
[9]
[10]
This research was supported by grants from Daewoo Pharmaceutical Co. Ltd. and National Research Foundation of Korea (2010-0003486).
[11]
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