Journal of Chromatography B, 998 (2015) 1–7
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Liquid chromatography–mass spectrometry analysis of five bisphosphonates in equine urine and plasma April S.Y. Wong a,∗ , Emmie N.M. Ho a , Terence S.M. Wan a,∗ , Kenneth K.H. Lam b , Brian D. Stewart b a b
Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China Department of Veterinary Regulation & International Liaison, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China
a r t i c l e
i n f o
Article history: Received 27 March 2015 Received in revised form 3 June 2015 Accepted 17 June 2015 Available online 20 June 2015 Keywords: Amino bisphosphonates Non-amino bisphosphonates Horse Plasma Urine Liquid chromatography–mass spectrometry Tiludronic acid Doping control analysis
a b s t r a c t Bisphosphonates are used in the management of skeletal disorder in humans and horses, with tiludronic acid being the first licensed veterinary medicine in the treatment of lameness associated with degenerative joint disease. Bisphosphonates are prohibited in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities). In order to control the use of bisphosphonates in equine sports, an effective method to detect the use of bisphosphonates is required. Bisphosphonates are difficult-to-detect drugs due to their hydrophilic properties. The complexity of equine matrices also added to their extraction difficulties. This study describes a method for the simultaneous detection of five bisphosphonates, namely alendronic acid, clodronic acid, ibandronic acid, risedronic acid and tiludronic acid, in equine urine and plasma. Bisphosphonates were first isolated from the sample matrices by solid-phase extractions, followed by methylation with trimethylsilyldiazomethane prior to liquid chromatography – tandem mass spectrometry analysis using selective reaction monitoring in the positive electrospray ionization mode. The five bisphosphonates could be detected at low ppb levels in 0.5 mL equine plasma or urine with acceptable precision, fast instrumental turnaround time, and negligible matrix interferences. The method has also been applied to the excretion study of tiludronic acid in plasma and urine collected from a horse having been administered a single dose of tiludronic acid. The applicability and effectiveness of the method was demonstrated by the successful detection and confirmation of the presence of tiludronic acid in an overseas equine urine sample. To our knowledge, this is the first reported method in the successful screening and confirmation of five amino- and non-amino bisphosphonates in equine biological samples. © 2015 Elsevier B.V. All rights reserved.
1. Background Bisphosphonates are in a class of compound characterized by the −C(PO3 )2 group acting as effective bone resorption inhibitors. These substances are used extensively in the management of skeletal disorders. Bisphosphonates are categorized as non-nitrogen-containing (e.g. tiludronic acid, clodronic acid) and nitrogen containing bisphosphonates (e.g. alendronic acid, risedronic acid, ibandronic acid) and have different mechanisms of action to inhibit osteoclast-mediated bone resorption [1]. They all share a common P–C–P moiety where the two phosphate groups are covalently linked to a carbon atom. Pharmacological proper-
∗ Corresponding authors. Fax: +86 852 2601 6564. E-mail addresses: [email protected] (A.S.Y. Wong), [email protected] (T.S.M. Wan). http://dx.doi.org/10.1016/j.jchromb.2015.06.020 1570-0232/© 2015 Elsevier B.V. All rights reserved.
ties, such as pharmacokinetics and drug strength are influenced by the chemical structures of the other functional groups (Fig. 1). They have been used clinically in human medicine for the treatment of osteoporosis, Paget’s disease, malignant diseases, and in hypercalcaemia to reduce skeletal morbidity associated with bone metastases. Bisphosphonates were introduced, with tiludronic acid being the first licensed in Europe in 2011, as a veterinary medicine in the treatment of equine orthopaedic conditions [2–4]. Under the rules of racing in many countries, the presence of bisphosphonates is prohibited in samples taken from racehorses. However, a lack of understanding of their pharmacokinetics in horses has prevented the proper control of their use. A sensitive analytical method is therefore essential for elucidating their elimination profile in horses. A number of analytical methods have been reported for the detection of bisphosphonates in human plasma and serum [5–13]; human urine [5,7,10,14–16], and equine plasma [17]. The develop-
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O P
Cl Cl
P O
O
OH
P
OH
Cl
OH
S P
OH
O
(a)
OH OH OH OH
(b)
O H 2N
P
HO
P
O
OH OH OH
H 3C
OH
O (c)
P
HO N CH3
P O
OH OH OH OH
(d)
O
N
P
HO
P O
OH OH OH OH
(e) Fig. 1. Chemical structures of (a) clodronic acid, and (b) tiludronic acid in the non-nitrogen containing bisphosphonates group; and (c) alendronic acid, (d) ibandronic acid, and (e) risedronic acid in the nitrogen containing bisphosphonate group.
ment of analytical method for the screening of bisphosphonates in biological matrices is challenging due to the chemical properties of these compounds. Bisphosphonates are hydrophilic compounds and ionize extensively in aqueous solution thus making simple liquid/liquid extraction ineffective. Ion-pair extraction using solid-phase extraction with 1-octyltriethylammonium phosphate as the counter ion [15] and liquid/liquid extraction using tetrabutylammonium bromide in chloroform [18] have been reported. Another widely-used but rather tedious sample extraction procedure is to isolate bisphosphonates from human matrix by repeated co-precipitation of bisphosphonates with calcium salts under alkaline condition, followed by sample clean-up or derivatization [5,7,14,18–20]. Due to the lack of chromophores in bisphoshonates, spectrophotometric detection after chromatographic separation is usually achieved using UV or fluorescent active derivatizating reagents (alendronate with 9-fluorenylmethy chloroformate [9,14]; olpadronate with 9-fluorenylmethy chloroformate [7]; alendronate with 2,3-naphthalene dicarboxyaldehyde [5], and pamidronate with 1-naphthylisothiocyante [20]. Bisphosphonates are poorly retained in reversed phase analytical columns and one approach to providing adequate retention is by employing ion-pair chromatography with the addition of an ion-pair reagent during sample preparation. Due to the difficulties mentioned above, a wide range of analytical techniques, such as gas chromatography [21], ion chromatography [22], capillary electrophoresis–electrospray ionization mass spectrometry [23], inductively coupled plasma mass spectrometry [24], and evaporative light-scattering detection with liquid chromatography [25] have been used to try to overcome the problems in detecting bisphosphonates in human biological matrices. Simultaneous detection of different bisphosphonates is seldom reported due to the difficulty in optimizing condition for the separation and detection of bisphosphonates on the reversed phase columns. This paper describes a liquid chromatography–mass spectrometry method which is capable of detecting five bisphosphonates in equine urine and plasma at ppb levels. To the best of our knowl-
edge, this is the first reported method for the detection of multiple bisphosphonates in equine matrixes. This method has been successfully applied to the detection and quantification of tiludronic acid in both post-administration equine urine and blood samples. 2. Experimental 2.1. Chemicals and reagent Reference standards of bisphosphonates are purchased, respectively, and the sources are as listed. Alendronate sodium was obtained from Apotex Inc. (Ontario, Canada). Clodronate disodium was from Schering (Berlin, Germany). Ibandronic sodium monohydrate was from Roche (New Jersey, USA). Risedronate sodium was from OSG Norwich Pharmaceuticals, Inc. (New York, USA). Tiludronic acid was from Ceva Sante Animale (Libourne, France). Ammonia solution was purchased from Merck (Darmstadt, Germany). Formic acid (>98%, RDH), methanol, trimethylsilyldiazomethane solution (2.0 M in diethyl ether) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Oasis® HLB cartridge (60 mg, 3 mL) and Oasis® WAX cartridge (60 mg, 3 mL) were purchased from Waters Corporation (Massachusetts, USA). Deionized water was generated from an in-house water purification system (Milli-Q, Molsheim, France). 2.2. Sample preparation and extraction procedures Urine and blood samples were centrifuged at 3000 rpm for 10 min. A portion of the plasma or urine (0.5 mL) was diluted to 3 mL with deionized water and adjusted to pH 4 with hydrochloric acid. The sample was filtered through an Oasis® HLB cartridge that had been pre-conditioned with methanol (3 mL) and deionized water (3 mL). The filtrate was adjusted to pH 4 with hydrochloric acid and loaded on an Oasis® WAX cartridge that had been pre-conditioned with methanol (2 mL) and formic acid (formic acid in deionized water, pH 4; 2 mL). The cartridge was then washed with formic
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Table 1 Method validation results for bisphosphonates spiked in equine urine. Target drug
Alendronic acid Clodronic acid Ibandronic acid Risedronic acid Tiludronic acid
Limit of detection (ng/mL)
100 50 25 50 10
QC level (ng/mL)
1000 500 250 500 100
Recovery (%)
31.3 86.5 55.0 49.7 34.3
Intra-day precision (% RSD; n = 5)a
Inter-day precision (% RSD; n = 5)a
PAR
RRT
PAR
RRT
12.21 11.92 8.56 12.03 12.23
0.00 0.00 0.00 0.01 0.01
15.23 12.32 18.73 18.42 18.27
0.01 0.01 0.01 0.26 0.26
a Risedronic acid was used as the internal standard for other targets in determining the peak area ratios (PAR) and the relative retention times (RRT). Tiludronic acid was used as the internal standard for risedronic acid in determining its PAR and RRT.
Table 2 Method validation results for bisphosphonates spiked in equine plasma. Target drug
Alendronic acid Clodronic acid Ibandronic acid Risedronic acid Tiludronic acid
Limit of detection (ng/mL)
100 50 25 50 10
QC level (ng/mL)
1000 500 250 500 100
Recovery (%)
30.9 41.4 16.9 30.1 22.5
Intra-day precision (% RSD; n = 5)a
Inter-day precision (% RSD; n = 5)a
PAR
RRT
PAR
RRT
8.01 16.59 4.46 13.46 14.43
0.00 0.00 0.00 0.01 0.01
18.90 35.61 12.72 29.85 23.74
0.45 1.50 0.46 0.48 0.48
a Risedronic acid was used as the internal standard for other targets in determining the peak area ratios (PAR) and the relative retention times (RRT). Tiludronic acid was used as the internal standard for risedronic acid in determining its PAR and RRT.
acid (formic acid in deionized water, pH 4; 2 mL) and methanol (2 mL), and then eluted with ammonia solution (10% ammonia in methanol). The eluate was evaporated to dryness under nitrogen at 60 ◦ C, reconstituted in deionized water (50 L), and methylated using trimethylsilyldiazomethane (100 L) and methanol (100 L). The solution was incubated at ambient temperature for 1 h and was transferred to a conical insert in a Chrompack autosampler vial for LC/SRM analysis.
the selection of the precursor and the corresponding product ions in Q1 and Q3 were both at 0.7 amu (FWHM). The scan width for product ions was set at 0.01 amu and the scan time was at 50 ms. The collision-induced dissociation (CID) energies were set at 5 V at the source and the collision energies ranged from 20 to 40 eV for the collision cell. Argon was used as the collision gas and was set at 1.2 mTorr. Data processing was performed using Thermo Finnigan XCalibur software (Version 2.0.7) (Thermo Fisher Scientific Inc., San Jose, USA).
2.3. Instrumentation 2.6. Semi-quantification of target compounds LC/SRM analyses were performed on a Thermo Finnigan TSQ Ultra mass spectrometer (Thermo Finnigan, San Jose, CA USA) with the electrospray source interfaced with a Waters UPLC system consisting of a binary gradient pump and an autosampler. Solid phase extraction (SPE) was carried out using a CEREXTM SPE Processor (Varian, Walnut Creek, CA, USA). 2.4. LC conditions for the screening of bisphosphonates A reversed-phase Waters XBridge C18 (15 cm × 2.1 mm ID; 3.5 m particle size, Waters, Milford, MA, USA) was used for the separation of the target analytes. The mobile phase was composed of 0.1% formic acid in deionised water as solvent A and 0.1% formic acid in acetonitrile as solvent B. A linear gradient was run at a flow rate of 200 L/min, with 98% solvent A and 2% solvent B at initial condition (t = 0 min), decreasing linearly to 0% solvent A from t = 1 min to t = 10 min, and held for 4 min (until t = 14 min). The gradient was then returned to 98% solvent A and 2% solvent B from t = 14 min to t = 15 min, and stabilised until t = 20 min before the next injection. The injection volume was 5 L. 2.5. MS conditions for the screening of bisphosphonates Detection of the bisphosphonates was performed under positive electrospray ionization in SRM mode with a single time segment. A spray voltage of 2500 V and a capillary temperature of 320 ◦ C were employed. The nitrogen sheath and auxiliary gas flow rates were set at 50 arbitrary TSQ Quantum units. The peak widths for
For each batch of urine or plasma samples, a calibrator containing the targeted drugs spiked in negative horse matrix was processed in parallel. The spiked concentration of individual targets in the calibrator was five times higher than the corresponding quality control (QC) sample shown in Tables 1 and 2. A one-point calibration was set for each analyte using the Thermo Finnigan XCalibur (Version 2.0.7) software. The concentrations of the target analytes in urine or plasma samples were estimated automatically by the software. 2.7. Administration study of tiludronic acid A single dose of tiludronic acid (1.755 g in water) was administered intravenously to a thoroughbred gelding clinically diagnosed with arthritis. Blood samples were collected before administration, and 0.1, 0.5, 1, 2, 4, 8, 12, and 24 h post administration. Urine samples were collected before administration, and 0.2, 0.3, 0.5, 1.0, 1.1, 1.2, 2.0, 3.0 and 7.0 days post administration. The samples were stored at 4 ◦ C upon received. 2.8. Quantification of tiludronic acid in the post-administration equine urine and plasma samples The calibrators for tiludronic acid were prepared in duplicate at 0, 250, 500, 1000, 1500, 2000 and 2500 ng/mL in urine and plasma. The internal standard (IS) risedronic acid was fortified at 2000 ng/mL in both urine and plasma samples. The concentrations
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Fig. 2. Product-ion chromatograms of the targeted bisphosphonates obtained from the analysis of (a) urine sample, and (b) plasma sample spiked with bisphosphonates at the control level.
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3. Results and discussion
methylating agents for bisphosphonates. Trimethylorthoacetate (TMOA) was reported to have unsatisfactory derivatization yield for ibandronic acid in plasma samples [8], which is one of our intended drug targets. Trimethylsilyldiazomethane (TMSD), commercially available as a 2 M ether solution, was chosen for this study. It is compatible with aqueous media and is, therefore, a highly effective methylating agent for the water soluble bisphosphonates. During our method development, it was observed that the complexity of equine matrices play a key role in the method robustness and recovery of bisphosphonates. The method was initially established with sample preparation using 3 mL equine urine (or 3 mL equine plasma), and loaded directly, after pH adjustment, onto a pre-conditioned mixed-mode weak anion exchange cartridge (Oasis® WAX cartridge). This method, demonstrated acceptable precision and recovery. It was found that moderate recoveries were obtained for tiludronic acid in urine and alendronic acid in plasma, while low recoveries were observed for the majority of other bisphosphonates [29]. In spite of their low recoveries, these analytes could still be detected consistently by the method at their quality control levels. As matrix effect was postulated to be the major contribution factor to the low extraction recoveries observed, subsequent method development was performed using a smaller sample volume of equine urine or equine plasma (0.5 mL). In addition, a polymeric sorbent (Oasis® HLB cartridge) was added before the mixed-mode weak anion exchange cartridge for further clean-up. These method modifications greatly improved both the inter-day and intra-day precisions and recoveries of the bisphosphonates. For example, the recovery of alendronic acid in urine was improved from 1.59% to 31.3%, and the recovery of clodronic acid in urine was improved from 10.98% to 86.5%.
3.1. Sample preparation
3.2. Method characteristics
Bisphosphonates are hydrophilic compounds that are poorly retained on reversed phase sorbents. Methylation prior to LC/MS analysis is a viable technique to reduce the polarity of the bisphosphonates before column chromatography. Derivatizing agents diazomethane [10], trimethylsilyldiazomethane [8,10,16,26,27] and trimethylorthoacetate [17,28] had been reported to be effective
Fig. 2a and b shows the product-ion chromatograms of the five bisphosphonates from spiked equine urine and plasma samples, respectively, at the QC Levels (Tables 1 and 2). All targeted bisphosphonates could be easily detected. At least 2 SRM transitions were monitored for each bisphosphonate. Method validation results for bisphosphonates in equine urine and plasma are summarized in
Fig. 3. Urinary excretion profile of tiludronic acid after single dose of 1.755 g tiludronic acid intravenous administration.
of tiludronic acid in post-administration samples were determined by interpolation from the calibration curves. QC samples (comprising negative urine and plasma samples each fortified with 1000 ng/mL of tiludronic acid) were analysed in duplicate for each batch of samples to verify that the analysis was in control. Samples with concentrations above the calibration range were diluted with a blank matrix before sample extraction. For quantitative analysis of the administration samples, data acquisition was performed in the SRM mode, monitoring transitions m/z 375 → 157 for tiludronic acid and m/z 340 → 182 for the IS, risedronic acid.
Fig. 4. Plasma elimination profile of tiludronic acid after single dose of 1.755 g tiludronic acid intravenous administration.
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Fig. 5. Confirmation of tiludronic acid in an overseas equine urine sample. Product-ion chromatograms of tiludronic acid from the analysis of (a) an overseas urine sample, and (b) a reference standard of a tiludronic acid.
Tables 1 and 2. Limits of detection were determined by analyzing spiked urine and plasma samples at different concentrations (equivalent to 10, 20 or 50% of the concentration in the QC sample). The limit of detection for a target analyte represents the lowest spiked concentration evaluated that gave a signal-to-noise ratio greater than 3:1 in the product-ion chromatogram. The method specificity was assessed using different blank equine urine (n = 80) and plasma (n = 80) samples. No significant interferences from the matrices at the retention times of the targeted ion transitions were observed. Method precision for the analyte-to-IS peak area ratios (PAR) and the relative retention times (RRT) were evaluated by replicate analyses (n = 5) of a spiked urine sample or a spiked plasma sample at their respective QC levels on 4 different days. Risedronic acid was used as the internal standard for other targets in determining the peak area ratios (PAR) and the relative retention times (RRT). Tiludronic acid was used as the internal standard for risedronic acid in determining its PAR and RRT. These results indicated that the methods have sufficiently acceptable precision to be used on a regular basis for qualitative identification. Extraction recovery was further investigated by analysing urine and plasma sample in duplicate, which had been spiked with the targeted analytes either before or after sample extraction. The concentrations of the target analytes in the recovery study were at 5 times their respective QC levels in urine or plasma. The peak area ratios of the targets obtained from samples spiked before extraction were compared with those obtained from blank extracts spiked with the same amount of analytes after extraction. The recoveries for the five bis-
phosphonates ranged from 31 to 87% in urine and 17 to 41% in plasma. 3.3. Method applicability This method was applied to an administration study of tiludronic acid for the treatment of a thoroughbred gelding clinically diagnosed with arthritis. Pre- and post-administration urine and blood samples were collected for analysis. Calibration graphs were linear over the range 0–2500 ng/ml for tiludronic acid in urine matrix (r2 > 0.9929; y = 0.9019x − 0.0307) and for tiludronic acid in plasma matrix (r2 > 0.9954; y = 0.5458x + 0.0197). Urinary tiludronic acid peaked at 13 h post administration, with the maximum concentration observed at about 23.0 g/mL (Fig. 3). Plasma tiludronic acid peaked at 0.1 h post-administration, with the maximum concentration observed at about 9.3 g/mL (Fig. 4). Tiludronic acid remained detectable for up to 7 days post-administration in urine, and up to 1 day post-administration in plasma, which were when the last samples had been collected. The excretion of tiludronic acid in urine was observed to be rather erratic as shown in Fig. 3. Similar observations were found in urine samples collected from horses during training which had been treated with tiludronic acid (unpublished results). One of the possible reasons might be that tiludronic acid is incorporated into the bone matrix from where it can be spontaneously released. Therefore plasma may be a better matrix to detect bisphosphonates in the control of their misuse in equine sports. Popot et al. [17] also concluded from their six-horse
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study that due to the strong matrix effect and erratic detection in equine urine, the quantification of tiludronic acid was performed in equine plasma. The applicability and effectiveness of this method in detecting the target bisphosphonates in real samples was demonstrated when tiludronic acid was detected in an equine urine sample from overseas. Fig. 5 shows the LC/SRM data from the subsequently confirmatory analysis on this sample. Both the retention time and relative abundances (based on 4 SRM transitions) of the substance in question matched well with the tiludronic acid standard and met our in-house match criteria for LC/SRM, which is based on the “AORC Guidelines for the Minimum Criteria for Identification by Chromatography and Mass Spectrometry” [30]. 4. Conclusions A versatile method, involving an extraction after a filtration step, derivatisation and LC/SRM analysis, was developed for the screening of five bisphosphonates in horse urine and plasma. Bisphosphonates are hydrophilic drugs that are difficult to extract from biological matrices and are poorly retained on reversed phase columns. We have overcome these problems by employing a mixed-mode weak anion exchange solid-phase extraction and by methylation of the bisphosphonates with trimethylsilyldiazomethane prior to LC/SRM analysis. The developed method has a fast instrumental turnaround time, acceptable precision and recovery, and negligible interference from sample matrices. As detection was with selective reaction monitoring, the above method could easily be extended to detect additional bisphosphonates. The applicability of the method was demonstrated by its effective screening and confirmation of tiludronic acid in an overseas equine urine sample, and by using it to analyse tiludronic acid post-administration samples. Acknowledgements The technical assistance from Ms. Shirley Tang, Mr. Dickson Lai, Ms. Coco Ng and Mr. M. Y. Lau are gratefully acknowledged. References [1] J. Green, Bisphosphonates: preclinical review, Oncologist 9 (2004) 3–13. [2] S.A. Soto, A.C. Barbara, Bisphosphonates: pharmacology and clinical approach to their use in equine osteoarticular diseases, J. Equine Vet. Sci 34 (2014) 727–737. [3] R.G.G. Russell, Bisphosphonates: the first 40 years, Bone 49 (2011) 2–19. [4] J.E. Nieto, O. Maher, S.D. Stanley, H.K. Knych, J.R. Snyder, Pharmacokinetics, pharmacodynamics, and safety of zoledronic acid in horses, Am. J. Vet. Res. 74 (2013) 550–556. [5] W.F. Kline, B.K. Matuszewski, Improved determination of the bisphosphonate alendronate in human plasma and urine by automated precolumn derivatization and high-performance liquid chromatography with fluorescence and electrochemical detection, J. Chromatogr. – Biomed. Appl. 583 (1992) 183–193. [6] H.J. Leis, G. Fauler, W. Windischhofer, Use of 18O3 -clodronate as an internal standard for the quantitative analysis of clodronate in human plasma by gas chromatography/electron ionisation mass spectrometry, Rapid Commun. Mass Spectrom. 18 (2004) 2781–2784. [7] R.W. Sparidans, J. Den Hartigh, S. Cremers, J.H. Beijnen, P. Vermeij, Semi-automatic liquid chromatographic analysis of olpadronate in urine and serum using derivatization with (9-fluorenylmethyl) chloroformate, J. Chromatogr. B: Biomed. Sci. Appl. 738 (2000) 331–341. [8] I. Tarcomnicu, M.C. Gheorghe, L. Silvestro, S.R. Savu, I. Boaru, A. Tudoroniu, High-throughput HPLC–MS/MS method to determine ibandronate in human plasma for pharmacokinetic applications, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 877 (2009) 3159–3168. [9] M.-H. Yun, K. Kwon, High-performance liquid chromatography method for determining alendronate sodium in human plasma by detecting fluorescence: application to a pharmacokinetic study in humans, J. Pharm. Biomed. Anal. 40 (2006) 168–172.
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Liquid chromatography–mass spectrometry analysis of five bisphosphonates in equine urine and plasma April S.Y. Wong a,∗ , Emmie N.M. Ho a , Terence S.M. Wan a,∗ , Kenneth K.H. Lam b , Brian D. Stewart b a b
Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China Department of Veterinary Regulation & International Liaison, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China
a r t i c l e
i n f o
Article history: Received 27 March 2015 Received in revised form 3 June 2015 Accepted 17 June 2015 Available online 20 June 2015 Keywords: Amino bisphosphonates Non-amino bisphosphonates Horse Plasma Urine Liquid chromatography–mass spectrometry Tiludronic acid Doping control analysis
a b s t r a c t Bisphosphonates are used in the management of skeletal disorder in humans and horses, with tiludronic acid being the first licensed veterinary medicine in the treatment of lameness associated with degenerative joint disease. Bisphosphonates are prohibited in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities). In order to control the use of bisphosphonates in equine sports, an effective method to detect the use of bisphosphonates is required. Bisphosphonates are difficult-to-detect drugs due to their hydrophilic properties. The complexity of equine matrices also added to their extraction difficulties. This study describes a method for the simultaneous detection of five bisphosphonates, namely alendronic acid, clodronic acid, ibandronic acid, risedronic acid and tiludronic acid, in equine urine and plasma. Bisphosphonates were first isolated from the sample matrices by solid-phase extractions, followed by methylation with trimethylsilyldiazomethane prior to liquid chromatography – tandem mass spectrometry analysis using selective reaction monitoring in the positive electrospray ionization mode. The five bisphosphonates could be detected at low ppb levels in 0.5 mL equine plasma or urine with acceptable precision, fast instrumental turnaround time, and negligible matrix interferences. The method has also been applied to the excretion study of tiludronic acid in plasma and urine collected from a horse having been administered a single dose of tiludronic acid. The applicability and effectiveness of the method was demonstrated by the successful detection and confirmation of the presence of tiludronic acid in an overseas equine urine sample. To our knowledge, this is the first reported method in the successful screening and confirmation of five amino- and non-amino bisphosphonates in equine biological samples. © 2015 Elsevier B.V. All rights reserved.
1. Background Bisphosphonates are in a class of compound characterized by the −C(PO3 )2 group acting as effective bone resorption inhibitors. These substances are used extensively in the management of skeletal disorders. Bisphosphonates are categorized as non-nitrogen-containing (e.g. tiludronic acid, clodronic acid) and nitrogen containing bisphosphonates (e.g. alendronic acid, risedronic acid, ibandronic acid) and have different mechanisms of action to inhibit osteoclast-mediated bone resorption [1]. They all share a common P–C–P moiety where the two phosphate groups are covalently linked to a carbon atom. Pharmacological proper-
∗ Corresponding authors. Fax: +86 852 2601 6564. E-mail addresses: [email protected] (A.S.Y. Wong), [email protected] (T.S.M. Wan). http://dx.doi.org/10.1016/j.jchromb.2015.06.020 1570-0232/© 2015 Elsevier B.V. All rights reserved.
ties, such as pharmacokinetics and drug strength are influenced by the chemical structures of the other functional groups (Fig. 1). They have been used clinically in human medicine for the treatment of osteoporosis, Paget’s disease, malignant diseases, and in hypercalcaemia to reduce skeletal morbidity associated with bone metastases. Bisphosphonates were introduced, with tiludronic acid being the first licensed in Europe in 2011, as a veterinary medicine in the treatment of equine orthopaedic conditions [2–4]. Under the rules of racing in many countries, the presence of bisphosphonates is prohibited in samples taken from racehorses. However, a lack of understanding of their pharmacokinetics in horses has prevented the proper control of their use. A sensitive analytical method is therefore essential for elucidating their elimination profile in horses. A number of analytical methods have been reported for the detection of bisphosphonates in human plasma and serum [5–13]; human urine [5,7,10,14–16], and equine plasma [17]. The develop-
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O P
Cl Cl
P O
O
OH
P
OH
Cl
OH
S P
OH
O
(a)
OH OH OH OH
(b)
O H 2N
P
HO
P
O
OH OH OH
H 3C
OH
O (c)
P
HO N CH3
P O
OH OH OH OH
(d)
O
N
P
HO
P O
OH OH OH OH
(e) Fig. 1. Chemical structures of (a) clodronic acid, and (b) tiludronic acid in the non-nitrogen containing bisphosphonates group; and (c) alendronic acid, (d) ibandronic acid, and (e) risedronic acid in the nitrogen containing bisphosphonate group.
ment of analytical method for the screening of bisphosphonates in biological matrices is challenging due to the chemical properties of these compounds. Bisphosphonates are hydrophilic compounds and ionize extensively in aqueous solution thus making simple liquid/liquid extraction ineffective. Ion-pair extraction using solid-phase extraction with 1-octyltriethylammonium phosphate as the counter ion [15] and liquid/liquid extraction using tetrabutylammonium bromide in chloroform [18] have been reported. Another widely-used but rather tedious sample extraction procedure is to isolate bisphosphonates from human matrix by repeated co-precipitation of bisphosphonates with calcium salts under alkaline condition, followed by sample clean-up or derivatization [5,7,14,18–20]. Due to the lack of chromophores in bisphoshonates, spectrophotometric detection after chromatographic separation is usually achieved using UV or fluorescent active derivatizating reagents (alendronate with 9-fluorenylmethy chloroformate [9,14]; olpadronate with 9-fluorenylmethy chloroformate [7]; alendronate with 2,3-naphthalene dicarboxyaldehyde [5], and pamidronate with 1-naphthylisothiocyante [20]. Bisphosphonates are poorly retained in reversed phase analytical columns and one approach to providing adequate retention is by employing ion-pair chromatography with the addition of an ion-pair reagent during sample preparation. Due to the difficulties mentioned above, a wide range of analytical techniques, such as gas chromatography [21], ion chromatography [22], capillary electrophoresis–electrospray ionization mass spectrometry [23], inductively coupled plasma mass spectrometry [24], and evaporative light-scattering detection with liquid chromatography [25] have been used to try to overcome the problems in detecting bisphosphonates in human biological matrices. Simultaneous detection of different bisphosphonates is seldom reported due to the difficulty in optimizing condition for the separation and detection of bisphosphonates on the reversed phase columns. This paper describes a liquid chromatography–mass spectrometry method which is capable of detecting five bisphosphonates in equine urine and plasma at ppb levels. To the best of our knowl-
edge, this is the first reported method for the detection of multiple bisphosphonates in equine matrixes. This method has been successfully applied to the detection and quantification of tiludronic acid in both post-administration equine urine and blood samples. 2. Experimental 2.1. Chemicals and reagent Reference standards of bisphosphonates are purchased, respectively, and the sources are as listed. Alendronate sodium was obtained from Apotex Inc. (Ontario, Canada). Clodronate disodium was from Schering (Berlin, Germany). Ibandronic sodium monohydrate was from Roche (New Jersey, USA). Risedronate sodium was from OSG Norwich Pharmaceuticals, Inc. (New York, USA). Tiludronic acid was from Ceva Sante Animale (Libourne, France). Ammonia solution was purchased from Merck (Darmstadt, Germany). Formic acid (>98%, RDH), methanol, trimethylsilyldiazomethane solution (2.0 M in diethyl ether) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Oasis® HLB cartridge (60 mg, 3 mL) and Oasis® WAX cartridge (60 mg, 3 mL) were purchased from Waters Corporation (Massachusetts, USA). Deionized water was generated from an in-house water purification system (Milli-Q, Molsheim, France). 2.2. Sample preparation and extraction procedures Urine and blood samples were centrifuged at 3000 rpm for 10 min. A portion of the plasma or urine (0.5 mL) was diluted to 3 mL with deionized water and adjusted to pH 4 with hydrochloric acid. The sample was filtered through an Oasis® HLB cartridge that had been pre-conditioned with methanol (3 mL) and deionized water (3 mL). The filtrate was adjusted to pH 4 with hydrochloric acid and loaded on an Oasis® WAX cartridge that had been pre-conditioned with methanol (2 mL) and formic acid (formic acid in deionized water, pH 4; 2 mL). The cartridge was then washed with formic
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Table 1 Method validation results for bisphosphonates spiked in equine urine. Target drug
Alendronic acid Clodronic acid Ibandronic acid Risedronic acid Tiludronic acid
Limit of detection (ng/mL)
100 50 25 50 10
QC level (ng/mL)
1000 500 250 500 100
Recovery (%)
31.3 86.5 55.0 49.7 34.3
Intra-day precision (% RSD; n = 5)a
Inter-day precision (% RSD; n = 5)a
PAR
RRT
PAR
RRT
12.21 11.92 8.56 12.03 12.23
0.00 0.00 0.00 0.01 0.01
15.23 12.32 18.73 18.42 18.27
0.01 0.01 0.01 0.26 0.26
a Risedronic acid was used as the internal standard for other targets in determining the peak area ratios (PAR) and the relative retention times (RRT). Tiludronic acid was used as the internal standard for risedronic acid in determining its PAR and RRT.
Table 2 Method validation results for bisphosphonates spiked in equine plasma. Target drug
Alendronic acid Clodronic acid Ibandronic acid Risedronic acid Tiludronic acid
Limit of detection (ng/mL)
100 50 25 50 10
QC level (ng/mL)
1000 500 250 500 100
Recovery (%)
30.9 41.4 16.9 30.1 22.5
Intra-day precision (% RSD; n = 5)a
Inter-day precision (% RSD; n = 5)a
PAR
RRT
PAR
RRT
8.01 16.59 4.46 13.46 14.43
0.00 0.00 0.00 0.01 0.01
18.90 35.61 12.72 29.85 23.74
0.45 1.50 0.46 0.48 0.48
a Risedronic acid was used as the internal standard for other targets in determining the peak area ratios (PAR) and the relative retention times (RRT). Tiludronic acid was used as the internal standard for risedronic acid in determining its PAR and RRT.
acid (formic acid in deionized water, pH 4; 2 mL) and methanol (2 mL), and then eluted with ammonia solution (10% ammonia in methanol). The eluate was evaporated to dryness under nitrogen at 60 ◦ C, reconstituted in deionized water (50 L), and methylated using trimethylsilyldiazomethane (100 L) and methanol (100 L). The solution was incubated at ambient temperature for 1 h and was transferred to a conical insert in a Chrompack autosampler vial for LC/SRM analysis.
the selection of the precursor and the corresponding product ions in Q1 and Q3 were both at 0.7 amu (FWHM). The scan width for product ions was set at 0.01 amu and the scan time was at 50 ms. The collision-induced dissociation (CID) energies were set at 5 V at the source and the collision energies ranged from 20 to 40 eV for the collision cell. Argon was used as the collision gas and was set at 1.2 mTorr. Data processing was performed using Thermo Finnigan XCalibur software (Version 2.0.7) (Thermo Fisher Scientific Inc., San Jose, USA).
2.3. Instrumentation 2.6. Semi-quantification of target compounds LC/SRM analyses were performed on a Thermo Finnigan TSQ Ultra mass spectrometer (Thermo Finnigan, San Jose, CA USA) with the electrospray source interfaced with a Waters UPLC system consisting of a binary gradient pump and an autosampler. Solid phase extraction (SPE) was carried out using a CEREXTM SPE Processor (Varian, Walnut Creek, CA, USA). 2.4. LC conditions for the screening of bisphosphonates A reversed-phase Waters XBridge C18 (15 cm × 2.1 mm ID; 3.5 m particle size, Waters, Milford, MA, USA) was used for the separation of the target analytes. The mobile phase was composed of 0.1% formic acid in deionised water as solvent A and 0.1% formic acid in acetonitrile as solvent B. A linear gradient was run at a flow rate of 200 L/min, with 98% solvent A and 2% solvent B at initial condition (t = 0 min), decreasing linearly to 0% solvent A from t = 1 min to t = 10 min, and held for 4 min (until t = 14 min). The gradient was then returned to 98% solvent A and 2% solvent B from t = 14 min to t = 15 min, and stabilised until t = 20 min before the next injection. The injection volume was 5 L. 2.5. MS conditions for the screening of bisphosphonates Detection of the bisphosphonates was performed under positive electrospray ionization in SRM mode with a single time segment. A spray voltage of 2500 V and a capillary temperature of 320 ◦ C were employed. The nitrogen sheath and auxiliary gas flow rates were set at 50 arbitrary TSQ Quantum units. The peak widths for
For each batch of urine or plasma samples, a calibrator containing the targeted drugs spiked in negative horse matrix was processed in parallel. The spiked concentration of individual targets in the calibrator was five times higher than the corresponding quality control (QC) sample shown in Tables 1 and 2. A one-point calibration was set for each analyte using the Thermo Finnigan XCalibur (Version 2.0.7) software. The concentrations of the target analytes in urine or plasma samples were estimated automatically by the software. 2.7. Administration study of tiludronic acid A single dose of tiludronic acid (1.755 g in water) was administered intravenously to a thoroughbred gelding clinically diagnosed with arthritis. Blood samples were collected before administration, and 0.1, 0.5, 1, 2, 4, 8, 12, and 24 h post administration. Urine samples were collected before administration, and 0.2, 0.3, 0.5, 1.0, 1.1, 1.2, 2.0, 3.0 and 7.0 days post administration. The samples were stored at 4 ◦ C upon received. 2.8. Quantification of tiludronic acid in the post-administration equine urine and plasma samples The calibrators for tiludronic acid were prepared in duplicate at 0, 250, 500, 1000, 1500, 2000 and 2500 ng/mL in urine and plasma. The internal standard (IS) risedronic acid was fortified at 2000 ng/mL in both urine and plasma samples. The concentrations
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Fig. 2. Product-ion chromatograms of the targeted bisphosphonates obtained from the analysis of (a) urine sample, and (b) plasma sample spiked with bisphosphonates at the control level.
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3. Results and discussion
methylating agents for bisphosphonates. Trimethylorthoacetate (TMOA) was reported to have unsatisfactory derivatization yield for ibandronic acid in plasma samples [8], which is one of our intended drug targets. Trimethylsilyldiazomethane (TMSD), commercially available as a 2 M ether solution, was chosen for this study. It is compatible with aqueous media and is, therefore, a highly effective methylating agent for the water soluble bisphosphonates. During our method development, it was observed that the complexity of equine matrices play a key role in the method robustness and recovery of bisphosphonates. The method was initially established with sample preparation using 3 mL equine urine (or 3 mL equine plasma), and loaded directly, after pH adjustment, onto a pre-conditioned mixed-mode weak anion exchange cartridge (Oasis® WAX cartridge). This method, demonstrated acceptable precision and recovery. It was found that moderate recoveries were obtained for tiludronic acid in urine and alendronic acid in plasma, while low recoveries were observed for the majority of other bisphosphonates [29]. In spite of their low recoveries, these analytes could still be detected consistently by the method at their quality control levels. As matrix effect was postulated to be the major contribution factor to the low extraction recoveries observed, subsequent method development was performed using a smaller sample volume of equine urine or equine plasma (0.5 mL). In addition, a polymeric sorbent (Oasis® HLB cartridge) was added before the mixed-mode weak anion exchange cartridge for further clean-up. These method modifications greatly improved both the inter-day and intra-day precisions and recoveries of the bisphosphonates. For example, the recovery of alendronic acid in urine was improved from 1.59% to 31.3%, and the recovery of clodronic acid in urine was improved from 10.98% to 86.5%.
3.1. Sample preparation
3.2. Method characteristics
Bisphosphonates are hydrophilic compounds that are poorly retained on reversed phase sorbents. Methylation prior to LC/MS analysis is a viable technique to reduce the polarity of the bisphosphonates before column chromatography. Derivatizing agents diazomethane [10], trimethylsilyldiazomethane [8,10,16,26,27] and trimethylorthoacetate [17,28] had been reported to be effective
Fig. 2a and b shows the product-ion chromatograms of the five bisphosphonates from spiked equine urine and plasma samples, respectively, at the QC Levels (Tables 1 and 2). All targeted bisphosphonates could be easily detected. At least 2 SRM transitions were monitored for each bisphosphonate. Method validation results for bisphosphonates in equine urine and plasma are summarized in
Fig. 3. Urinary excretion profile of tiludronic acid after single dose of 1.755 g tiludronic acid intravenous administration.
of tiludronic acid in post-administration samples were determined by interpolation from the calibration curves. QC samples (comprising negative urine and plasma samples each fortified with 1000 ng/mL of tiludronic acid) were analysed in duplicate for each batch of samples to verify that the analysis was in control. Samples with concentrations above the calibration range were diluted with a blank matrix before sample extraction. For quantitative analysis of the administration samples, data acquisition was performed in the SRM mode, monitoring transitions m/z 375 → 157 for tiludronic acid and m/z 340 → 182 for the IS, risedronic acid.
Fig. 4. Plasma elimination profile of tiludronic acid after single dose of 1.755 g tiludronic acid intravenous administration.
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Fig. 5. Confirmation of tiludronic acid in an overseas equine urine sample. Product-ion chromatograms of tiludronic acid from the analysis of (a) an overseas urine sample, and (b) a reference standard of a tiludronic acid.
Tables 1 and 2. Limits of detection were determined by analyzing spiked urine and plasma samples at different concentrations (equivalent to 10, 20 or 50% of the concentration in the QC sample). The limit of detection for a target analyte represents the lowest spiked concentration evaluated that gave a signal-to-noise ratio greater than 3:1 in the product-ion chromatogram. The method specificity was assessed using different blank equine urine (n = 80) and plasma (n = 80) samples. No significant interferences from the matrices at the retention times of the targeted ion transitions were observed. Method precision for the analyte-to-IS peak area ratios (PAR) and the relative retention times (RRT) were evaluated by replicate analyses (n = 5) of a spiked urine sample or a spiked plasma sample at their respective QC levels on 4 different days. Risedronic acid was used as the internal standard for other targets in determining the peak area ratios (PAR) and the relative retention times (RRT). Tiludronic acid was used as the internal standard for risedronic acid in determining its PAR and RRT. These results indicated that the methods have sufficiently acceptable precision to be used on a regular basis for qualitative identification. Extraction recovery was further investigated by analysing urine and plasma sample in duplicate, which had been spiked with the targeted analytes either before or after sample extraction. The concentrations of the target analytes in the recovery study were at 5 times their respective QC levels in urine or plasma. The peak area ratios of the targets obtained from samples spiked before extraction were compared with those obtained from blank extracts spiked with the same amount of analytes after extraction. The recoveries for the five bis-
phosphonates ranged from 31 to 87% in urine and 17 to 41% in plasma. 3.3. Method applicability This method was applied to an administration study of tiludronic acid for the treatment of a thoroughbred gelding clinically diagnosed with arthritis. Pre- and post-administration urine and blood samples were collected for analysis. Calibration graphs were linear over the range 0–2500 ng/ml for tiludronic acid in urine matrix (r2 > 0.9929; y = 0.9019x − 0.0307) and for tiludronic acid in plasma matrix (r2 > 0.9954; y = 0.5458x + 0.0197). Urinary tiludronic acid peaked at 13 h post administration, with the maximum concentration observed at about 23.0 g/mL (Fig. 3). Plasma tiludronic acid peaked at 0.1 h post-administration, with the maximum concentration observed at about 9.3 g/mL (Fig. 4). Tiludronic acid remained detectable for up to 7 days post-administration in urine, and up to 1 day post-administration in plasma, which were when the last samples had been collected. The excretion of tiludronic acid in urine was observed to be rather erratic as shown in Fig. 3. Similar observations were found in urine samples collected from horses during training which had been treated with tiludronic acid (unpublished results). One of the possible reasons might be that tiludronic acid is incorporated into the bone matrix from where it can be spontaneously released. Therefore plasma may be a better matrix to detect bisphosphonates in the control of their misuse in equine sports. Popot et al. [17] also concluded from their six-horse
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study that due to the strong matrix effect and erratic detection in equine urine, the quantification of tiludronic acid was performed in equine plasma. The applicability and effectiveness of this method in detecting the target bisphosphonates in real samples was demonstrated when tiludronic acid was detected in an equine urine sample from overseas. Fig. 5 shows the LC/SRM data from the subsequently confirmatory analysis on this sample. Both the retention time and relative abundances (based on 4 SRM transitions) of the substance in question matched well with the tiludronic acid standard and met our in-house match criteria for LC/SRM, which is based on the “AORC Guidelines for the Minimum Criteria for Identification by Chromatography and Mass Spectrometry” [30]. 4. Conclusions A versatile method, involving an extraction after a filtration step, derivatisation and LC/SRM analysis, was developed for the screening of five bisphosphonates in horse urine and plasma. Bisphosphonates are hydrophilic drugs that are difficult to extract from biological matrices and are poorly retained on reversed phase columns. We have overcome these problems by employing a mixed-mode weak anion exchange solid-phase extraction and by methylation of the bisphosphonates with trimethylsilyldiazomethane prior to LC/SRM analysis. The developed method has a fast instrumental turnaround time, acceptable precision and recovery, and negligible interference from sample matrices. As detection was with selective reaction monitoring, the above method could easily be extended to detect additional bisphosphonates. The applicability of the method was demonstrated by its effective screening and confirmation of tiludronic acid in an overseas equine urine sample, and by using it to analyse tiludronic acid post-administration samples. Acknowledgements The technical assistance from Ms. Shirley Tang, Mr. Dickson Lai, Ms. Coco Ng and Mr. M. Y. Lau are gratefully acknowledged. References [1] J. Green, Bisphosphonates: preclinical review, Oncologist 9 (2004) 3–13. [2] S.A. Soto, A.C. Barbara, Bisphosphonates: pharmacology and clinical approach to their use in equine osteoarticular diseases, J. Equine Vet. Sci 34 (2014) 727–737. [3] R.G.G. Russell, Bisphosphonates: the first 40 years, Bone 49 (2011) 2–19. [4] J.E. Nieto, O. Maher, S.D. Stanley, H.K. Knych, J.R. Snyder, Pharmacokinetics, pharmacodynamics, and safety of zoledronic acid in horses, Am. J. Vet. Res. 74 (2013) 550–556. [5] W.F. Kline, B.K. Matuszewski, Improved determination of the bisphosphonate alendronate in human plasma and urine by automated precolumn derivatization and high-performance liquid chromatography with fluorescence and electrochemical detection, J. Chromatogr. – Biomed. Appl. 583 (1992) 183–193. [6] H.J. Leis, G. Fauler, W. Windischhofer, Use of 18O3 -clodronate as an internal standard for the quantitative analysis of clodronate in human plasma by gas chromatography/electron ionisation mass spectrometry, Rapid Commun. Mass Spectrom. 18 (2004) 2781–2784. [7] R.W. Sparidans, J. Den Hartigh, S. Cremers, J.H. Beijnen, P. Vermeij, Semi-automatic liquid chromatographic analysis of olpadronate in urine and serum using derivatization with (9-fluorenylmethyl) chloroformate, J. Chromatogr. B: Biomed. Sci. Appl. 738 (2000) 331–341. [8] I. Tarcomnicu, M.C. Gheorghe, L. Silvestro, S.R. Savu, I. Boaru, A. Tudoroniu, High-throughput HPLC–MS/MS method to determine ibandronate in human plasma for pharmacokinetic applications, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 877 (2009) 3159–3168. [9] M.-H. Yun, K. Kwon, High-performance liquid chromatography method for determining alendronate sodium in human plasma by detecting fluorescence: application to a pharmacokinetic study in humans, J. Pharm. Biomed. Anal. 40 (2006) 168–172.
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