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Rapid quantification of miglustat in human plasma and cerebrospinal fluid by liquid chromatography coupled with tandem mass spectrometry
Journal of Chromatography B, 877 (2009) 149–154
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Rapid quantification of miglustat in human plasma and cerebrospinal fluid by liquid chromatography coupled with tandem mass spectrometry Jérôme Guitton a,b,c,∗ , Sylvie Coste d , Nathalie Guffon-Fouilhoux c,e , Sabine Cohen d , Monique Manchon d , Marc Guillaumont d a
Laboratoire de ciblage thérapeutique en cancérologie, Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, F-69495 Pierre Bénite, France Laboratoire de Toxicologie, Université de Lyon 1, ISPB, Faculté de pharmacie, F-69008 Lyon, France Laboratoire Inserm U820 Métabolomique et maladies métaboliques, Faculté de médecine A. Carrel, Université de Lyon, F-69372 Lyon, France d Laboratoire de biochimie-toxicologie, Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, F-69495 Pierre Bénite, France e Service des Maladies Métaboliques, Hospices Civils de Lyon, Hôpital E. Herriot, F-69437 Lyon, France b c
a r t i c l e
i n f o
Article history: Received 24 July 2008 Accepted 26 November 2008 Available online 3 December 2008 Keywords: Miglustat OGT 918 Deoxynojirimycin Mass spectrometry Liquid chromatography
a b s t r a c t Miglustat (OGT 918) is an iminosugar recently introduced in therapeutic as potential alternative therapy in disorders found in several diseases such as Tay-Sachs, Gaucher or Niemann-Pick diseases. A highly sensitive liquid-chromatography–electrospray tandem mass spectrometry (LC–MS/MS) assay was developed for the quantification of miglustat in human plasma and cerebrospinal fluid (CSF). The sample preparation consists in a simple protein precipitation with a mixture of acetonitrile/methanol (75/25) which yields 100% recovery. The isocratic separation utilizes an Atlantis Hilic (3 m, 150 mm × 2.1 mm) column, with a mobile phase of acetonitrile/water/ammonium acetate buffer (75/10/15, v/v/v) delivered at 230 l/min. Selected reaction monitoring (SRM) mode was used with the transitions m/z 220 → 158 for the miglustat and m/z 208 → m/z 146 for the miglitol (internal standard). Good linearity was observed in a range from 125 to 2500 ng/ml and from 50 to 1000 ng/ml, for plasma and CSF, respectively. The within-run precision of the assay was less than 6%, and the between-run run precision was less than 6.5%, for six replicates at each of three concentrations and evaluated on three separated days for both plasma and CSF mediums. Assay accuracy was in the range of 98–106.5%. Stability of miglustat was reported under a variety of storage conditions. The miglustat concentrations in two children are presented to demonstrate the clinical interest of this new method. © 2008 Elsevier B.V. All rights reserved.
1. Introduction N-Butyl-1-deoxynojirimycin (OGT 918) is an iminosugar introduced in therapeutic with the name of miglustat (Zavesca) [1]. Miglustat is a competitive inhibitor of the ceramide-specific glucosyltransferase which catalyzes the first step in the synthesis of glycosphingolipids (GSLs), essential components of the eukaryote cell membranes [2]. Several genetic mutations may produce a pathogenic accumulation of GSLs into lysosomes leading to the GSLs storage disorders found in several diseases such as Tay-Sachs, Gaucher or Niemann-Pick diseases [3]. These diseases are characterized by visceral disorders and by progressive neuropathic malfunctions with important clinical heterogenic manifestations [4]. Unfortunately, until now, treatments were limited to supportive
∗ Corresponding author at: Laboratoire de ciblage thérapeutique en cancérologie, Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, F-69495 Pierre Bénite, France. E-mail address: [email protected] (J. Guitton). 1570-0232/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2008.11.040
care and management of intervening problems [5]. In this condition, miglustat has been recently proposed as a potential alternative therapy. As far as we know, only one assay has been reported for the analysis of N-alkylated iminosugars in biological samples [6]. This method, based on the high-performance cation exchange chromatography coupled to the pulsed amperometric detection, is time-consuming since compound extraction and purification require more than 2 h. Moreover no data in term of methodological validation was provided [6]. In one other study, the use of MALDI-ToF MS technique for the quantification of miglustat is only mentioned, but no analytical description was submitted [4]. The aim of the present study was to develop a simple and rapid assay for the quantification of miglustat from human plasma and cerebrospinal fluid (CSF). Due to the high polarity of miglustat a hydrophilic interaction liquid chromatography (Hilic) was selected, coupled with a quadrupole tandem mass spectrometry to enhance the selectivity of the method.
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Ionization suppression study was performed using a postcolumn infusion of 1 g/ml solution of miglustat and miglitol while a blank of plasma or CSF was injected. 2.4. Sample preparation
Fig. 1. Chemical structures of miglustat (N-butyl-1-deoxynojirimycin – MM: 219) (A) and miglitol (N-hydroxyethyl-1-deoxynojirimycin – MM: 207) (B).
2. Experimental 2.1. Reagents and chemicals The standard of miglustat was from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and was kindly furnished by Dr M-T Vanier (Inserm U820, France). Miglitol was from Pfizer (New York, USA). N-Methyldeoxynojirimycin (N-Methyl-dnj), reagent-grade ammonium acetate, ammonia (30%) were supplied from Sigma (St Quentin Falavier, France). HPLC-grade acetonitrile (ACN) was obtained from Merck (Darmstadt, Germany). Methanol was from Carlo Erba (Milano, Italy). The water used throughout the study was produced from a deionized water system Milli-Q (Millipore corporation, MA, USA). Drug free normal human plasma was provided by the regional blood bank (EFS rhône-Alpes, France). ReconstitutedCSF (R-CSF) preparation derived from a method proposed by Oka et al. and contains: glucose (0.8 g/l), albumin (0.2 g/l), NaCl (7.3 g/l), NaHCO3 (1.9 g/l), the mixture was adjusted at pH 7.4. All the reagents were supplied by Sigma [7]. Stock solutions of miglustat and miglitol were prepared in methanol at a concentration of 1 mg/ml, and stored at +4 ◦ C (see Fig. 1 for the structures). These solutions were then diluted with water to yield a series of spiking standard solutions. 2.2. Chromatographic conditions and instrumentation The chromatographic system used consisted of a Surveyor MS quaternary pump, Surveyor autosampler injector and column thermostater (ThermoElectron, San Jose, USA). HPLC isocratic separation of analytes was performed on a Waters Atlantis Hilic 3 m, 150 mm × 2.1 mm analytical column (Waters, Milford, USA) maintained at 25 ◦ C. The eluant was a mixture of acetonitrile/water/buffer (75/10/15, v/v/v) delivered at 230 l/min. The buffer consisted of ammonium acetate 100 mM, pH 5. 2.3. Mass spectrometric conditions and instrumentation The detector was a Quantum-Ultra (ThermoElectron, San Jose, USA) triple quadrupole mass spectrometer equipped with an IonMax API source. The instrument was operated in positive ion mode with electrospray (ESI) source. The position (x, y, z) of the ESI probe was optimized with miglustat. Argon was used as collision gas at 1.5 mTorr. Spray voltage and capillary temperature were set at 3.5 kV and 300 ◦ C, respectively. The pressures for the nitrogen sheath gas, auxiliary gas and sweep gas were maintained at 30, 10 and 5 units (units refer to an arbitrary value set by the X-calibur software). For the quantification of miglustat and the miglitol (internal standard), the transitions m/z 220 (Q1) → m/z 158 (Q3) (miglustat) and m/z 208 (Q1) → m/z 146 (Q3) (miglitol) were monitored using the selected reaction monitoring (SRM) with 500 ms dwell time per channel. The first (Q1) and the third (Q3) quadrupole were set with full-width at half maximum height of 0.7 Th. Collision energy was set at 24 eV and 22 eV for miglustat and miglitol, respectively.
All samples were prepared by simple protein precipitation. At 10 l of plasma was added 50 l of internal standard (75 g/ml), then 1 ml of mixture acetonitrile/methanol (75/25) was introduced. Samples were vortexed for 30 s, and centrifugation was performed at 13,000 × g for 10 min. For CSF, at 20 l of sample was added 20 l of internal standard (75 g/ml), then the preparation was the same as for plasma. At last, in both cases, 5 l of the supernatant was injected into the HPLC device by an autosampler maintained at +10 ◦ C. 2.5. Calibration curve and validation procedure 2.5.1. Calibration Calibration curves were prepared with blank plasma or R-CSF samples. Miglustat was added to plasma to yield final concentrations of 125, 250, 500, 1250, 2500 ng/ml and to R-CSF to yield final concentrations of 50, 100, 250, 500, 1000 ng/ml. Two clinical studies have shown that the plasma and the CSF concentrations were in these ranges of concentration when children were treated with miglustat for GSLs storage disorders diseases [1,5]. A total of 12 calibration curves in plasma and 5 in CSF were generated during the entire validation process. Peak area ratios of miglustat to the internal standard measured at each nominal concentration were used to construct weighted (1/concentration) least-square linear regression curves. 2.5.2. Precision and accuracy The between-run precision (BRP), the within-run precision (WRP) and accuracy of the method were evaluated on three separate days by six replicates analysis of quality control (QC) containing miglustat at concentration of 150, 750, 1500 ng/ml and 75, 375, 750 ng/ml for plasma and R-CSF, respectively. An estimate of the BRP was obtained by one-way analysis of variance (ANOVA) for each test concentration using “run day” as the classification variable. The WRP was determined as WRP = 100 ×
MSwit /GM . The BRP was estimated as BRP = 100 ×
(MSbet − MSwit )/n /GM . MSwit , MSbet , n, GM, represented
the within-groups mean square, the between-groups mean square, the number of replicate observations within each run and the grand mean, respectively. These parameters were calculated using the software Statview for windows version 5.0 (SAS institute, Cary, USA). The accuracy was expressed as a percentage of bias ((mean value − nominal value)/nominal value × 100). 2.5.3. Recovery, specificity and sensitivity The recovery was conducted at three concentrations (QC) by comparing the mean response of extracted spiked plasma samples, with those obtained from direct injection of the same amount dissolved in the mobile phase. The specificity of the method was tested by visual inspection of chromatograms of extracted human plasma samples from six donors for the presence of interfering peaks. The lower limit of quantification (LLOQ) was defined as the lowest standard concentration which produced assay result within ±20% of the nominal concentration, with a coefficient of variation less than 20%. 2.5.4. Stability Stability studies were conducted, under a variety of conditions, in blank plasma spiked at 150, 750, 1500 ng/ml (QC) in
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Fig. 2. MRM chromatograms for miglustat (m/z 220 → 158) (left) and miglitol (internal standard) (m/z 208 → 146) (right) in blank plasma (a), in R-CSF (b), in plasma spiked with miglustat at 125 ng/ml and miglitol at 3750 ng/ml (c); R-CSF spiked with miglustat at 50 ng/ml and miglitol at 1500 ng/ml (d).
duplicate and in R-CSF spiked at 75, 375, 750 ng/ml (QC) in duplicate. The auto-sampler stability (20 h at +10 ◦ C) of the compounds after extraction from spiked plasma was tested. The stability after storage of plasma and R-CSF aliquots at +4 ◦ C was performed by re-analyzing samples once a day for 3 days. The freeze–thaw stability of miglustat was tested following five and three cycles at −20 ◦ C in plasma and R-CSF, respectively. The samples were brought to room temperature, well vortexed, and extracted immediately. Furthermore, the long-term freezer stability at −20 ◦ C was tested with plasma samples from patients treated with miglustat and with spiked plasma samples and re-analyzed 6 months later the first analysis. At last, the stability of miglustat was checked at room temperature by analyzing samples every hour for 6 h.
2.6. Patient samples Plasma and CSF samples were obtained from two children treated by a daily miglustat dosage over 6 months. These patients were included in a study approved by an ethic committee. Each child and/or their parents gave written informed consent. Blood and CSF samples were collected on day 0 before treatment and 6 months later. Blood samples (5 ml) were collected in lithium heparin glass tubes and tubes were gently inverted several times. Then blood was centrifuged at 3000 × g for 10 min at +4 ◦ C, and plasma was transferred to 5 ml polypropylene tubes and kept at −20 ◦ C until analysis. CSF samples were collected in 5 ml glass tubes, centrifuged at 3000 × g for 10 min at +4 ◦ C and supernatants were kept at −20 ◦ C until analysis.
Fig. 3. Regions of ionization suppression in plasma and in CSF for miglustat (left) and miglitol (right). Chromatograms were obtained after injection of 5 l of blank plasma (a) and CSF (b) after extraction procedure. Experiment was carried out using a post-column infusion of a 1 g ml−1 solution (speed infusion: 5 l min−1 ). Arrow indicates the retention time of compounds.
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3. Results and discussion 3.1. Liquid chromatography During the method development, we spiked with miglustat three blank CSF samples from three patients untreated with miglustat. No difference was observed between these samples and R-CSF spiked at the same concentration. Consequently, we used a surrogate matrix for CSF throughout the assay. Both miglustat and miglitol eluted as sharp peaks and plasma, or CSF, matrix components did not interfere with the analysis (Fig. 2). With the chromatographic conditions described a retention times for miglustat (3.7 min) and miglitol (3.9 min) were found. Consequently, a short run-time of 5 min was achieved. Chromatographic separation performed with Hilic column allows to obtain peak shape, retention and ionization of high polar compounds such as miglustat and miglitol. This mode of chromatography has been used for many years for polar compounds as, for example sugars [8]. Ionization suppression regions were found at 1.0–2.0 min for the plasma for both compounds, and 2.0–2.5 min for the CSF in the case of miglustat (Fig. 3). The retention of miglustat and miglitol was adequate to avoid a modification of the signal. 3.2. Mass spectrometry N-Methyl-dnj was, in the first intention, selected as internal standard. However, this compound exhibited an important variation of response during a run. No explanation and no solution were found as satisfactory and miglitol was tested as internal standard. This compound did not exhibit this signal variation and was selected as internal standard. During the first step, due to the polarity of miglustat and miglitol, the ionization conditions were tuned by infusing a mixture of both products using the electrospray ion source (ESI). However, tests performed with an APCI source also showed a response roughly in the same range as the one obtained with ESI. In both cases, miglustat and miglitol formed predominantly protonated molecules ([M+H]+ ) in the mobile phase used in the present study. During the second step, optimal ionization conditions were set and ESI response was found to be more stable and slightly higher, consequently this source was selected for the rest of the assay. The most sensitive daughter ions for miglustat and miglitol were found at m/z 158 and 146, respectively (Fig. 4). The MS/MS parameters were optimized to maximize the response for miglustat parent/daughter ion of m/z 220 → 158 and for the miglitol of m/z 208 → 146 in the positive ion mode. Li and al. proposed a fragmentation pathway for the miglitol corresponding to the loss of the N-alkyl-chain and a dehydratation on the deoxynojirimycin ring (Fig. 5) [9]. Miglustat and miglitol are structural analogues, so, if this fragmentation pathway is to be retained, the production ion spectra for the two compounds should show similar fragmentation patterns with a predominant daughter ion at m/z 146. However, we observed two different main daughter ions at m/z 158 (miglustat) and m/z 146 (miglitol). For the two compounds, the mass spectra showed ions corresponding to a successive dehydratation (miglustat: 220, 202, 184, 166; miglitol: 208, 190, 172, 154) and a parent-main daughter ion difference equal to m/z 62 (Fig. 4). So, another fragmentation pathway has been proposed based on the opening of the deoxynojirimycin ring and no fragmentation on the N-alkyl-chain. The Fig. 6 presents the fragmentation proposed for miglustat. The fragmentation patters is the same for the miglitol with a mass shift corresponding to the N-alkylchain differences. Only labeled compounds on different positions should allow the thorough determination of the fragmentation patterns.
Fig. 4. Product ion mass spectrum (partial fragmentation) of miglustat (A) and miglitol (B).
3.3. Validation The calibration curves were obtained by weighted 1/[concentration] least-squares linear regression analysis of miglustat concentration versus peak area ratio of miglustat/miglitol area ratios. Standard curves exhibited excellent linearity in the range of 125–2500 ng/ml for plasma, and 50–1000 ng/ml for CSF, with coefficients of correlation greater than 0.996. The resulting assay precision and accuracy data are presented in Tables 1 and 2. Assay accuracy and precision are determined by the validation QC samples. The within-run precision of the assay was less than 6%, and the between-run run precision was less than 6.5%, for each concentration on three QC samples and for both plasma and CSF mediums. Assay accuracy was in the range of 98–106.5%. The lower limit of quantification of miglustat in both mediums was set at the lowest standard concentration. However, in order to evaluate the performance of this assay, plasma samples were also spiked at 25 ng/ml. For this concentration precision was within 20% and accu-
Fig. 5. Previous fragmentation pathway proposed for the miglitol [9].
J. Guitton et al. / J. Chromatogr. B 877 (2009) 149–154
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Fig. 6. Proposed fragmentation pathways for the miglustat leading to the main ions observed in the mass spectrum.
racy between 80 and 120%. The limit of quantification of the method could be easily set lower if necessary by using a volume sample higher or by reducing the volume of precipitation mixture. As suggested by Shah et al. the high QC should be near the upper boundary of the calibration curve [10]. In our study, this QC is between the upper limit of quantification and the next lowest point and could be set at higher concentration (2000 ng/ml for example). Since previous pharmacokinetic data indicated that upper plasma concentrations are mostly in the range 1000–1700 ng/ml, we choose to set the high QC at 1500 ng/ml. The extraction recovery of miglustat and miglitol was determined by comparing peak areas from plasma samples obtained after single step protein precipitation with those obtained from direct injections of standard solutions. The mean recovery of miglustat was 101, 102 and 102% for low, medium and high QC, respectively (n = 3). The mean recovery of miglitol was 101% (n = 9) which is the concentration used in plasma for the present study.
Table 1 Inter-day validation of the determination of miglustat in plasma and in cerebrospinal fluid (CSF). Concentration (ng/ml) Spiked
Precision (%) (between run)
Accuracy (%)
Found (mean ± S.D.) 10 15 30 51 33
7.8 6.3 5.9 4.2 1.3
100.8 98.1 102.3 98.0 100.8
CSF (n = 5) 50 100 250 500 1000
3.2 7 15 21 21
6.3 7.2 6.2 4.3 2.0
102.9 98.7 100.2 98.2 101.6
± ± ± ± ±
Stock solutions of miglustat and miglitol in methanol were stable for at least 6 months without observable degradation when stored at +4 ◦ C. After extraction from spiked plasma, the stability of miglustat and miglitol was demonstrated for at least 20 h when vials were maintained in the autosampler at +10 ◦ C. Consequently, the present method allows analyzing numerous samples within a single run. In spiked human plasma or R-CSF, miglustat was stable for at least 3 days when samples were stored at +4 ◦ C. In both mediums, miglustat is stable for at least 6 h at room temperature. In frozen human plasma and in R-CSF, miglustat has been found to be stable for 6 months at −20 ◦ C. Finally, no significant degradation (<10%) was observed for miglustat after five or three freeze–thaw cycles in plasma and R-CSF, respectively. This data is in agreement with the observations of Li et al. who showed that miglitol was stable under a variety of conditions [9]. 3.5. Application of the method The suitability of the developed method for clinical use was demonstrated by the determination of miglustat in plasma and CSF Table 2 Assessment of accuracy and precision in plasma and in cerebrospinal fluid (CSF).
Plasma (n = 12) 125 126 ± 250 245 ± 500 512 ± 1250 1224 ± 2500 2519 ± 51.5 99 250 491 1016
3.4. Stability
Data from calibration curves prepared as a single replicate and analyzed on different days.
Concentration (ng/ml)
Precision (%)
Accuracy (%)
Spiked
Found (mean ± S.D.)
Within-run
Between-run
Plasma 150 750 1500
160 ± 9 771 ± 43 1505 ± 92
5.5 3.4 2.9
1.7 5.3 6.4
106.5 102.9 100.4
CSF 75 375 750
75.7 ± 5.8 375 ± 17 735 ± 35
5.8 3.0 4.2
6.1 4.0 2.7
101.0 100.2 98.0
Data from six replicates for each concentration and analyzed on three different days.
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Fig. 7. MRM chromatograms for miglustat (m/z 220 → 158) (left) and miglitol (internal standard) (m/z 208 → 146) (right) in plasma from child before treatment (a); in CSF from child before treatment (b); and response in plasma (miglustat 1679 ng/ml) (c) and in CSF (miglustat 243 ng/ml) (d) for child treated over 6 months. Miglitol was present at 3750 ng/ml in plasma (a and c) and at 1500 ng/ml in CSF (b and d).
samples from two children treated by miglustat. The concentrations determined with both mediums were in agreement with previously published values [1,5]. No endogenous peaks were found interfering with miglustat or miglitol (Fig. 7). 4. Conclusion The new LC–MS/MS method described here provides major advantages in terms of simplicity of sample extraction, selectivity of detection and turnaround time as compared with previous HPLC method [6]. This method has been successfully applied to analyze miglustat concentrations in human plasma and CSF. This assay meets the requirements of bioanalytical methodology validation, providing good accuracy and precision. The present method permits the analysis of plasma and CSF human samples in a range of concentrations which are sufficiently sensitive to allow monitoring after oral administration of miglustat in GSLs diseases.
References [1] T. Cox, R. Lachmann, C. Hollak, J. Aerts, S. van Weely, M. Hrebicek, F. Platt, T. Butters, R. Dwek, C. Moyses, I. Gow, D. Elstein, A. Zimran, Lancet 355 (2000) 1481. [2] H.R. Mellor, J. Nolan, L. Pickering, M.R. Wormald, F.M. Platt, R.A. Dwek, G.W.J. Fleet, T.D. Butters, Biochem. J. 366 (2002) 225. [3] F.M. Platt, G.R. Neises, G. Reinkensmeier, M.J. Townsend, V.H. Perry, R.L. Proia, B. Winchester, R.A. Dwek, T.D. Butters, Science 276 (1997) 428. [4] F.M. Platt, G. Reinkensmeier, R.A. Dwek, T.D. Butters, J. Biol. Chem. 272 (1997) 19365. [5] B. Bembi, F. Marchetti, V.I. Guerci, G. Ciana, R. Addobbati, D. Grasso, R. Barone, R. Cariati, L. Fernandez-Guillen, T. Butters, M.G. Pittis, Neurology 66 (2006) 278. [6] H.R. Mellor, A. Adam, F.M. Platt, R.A. Dwek, T.D. Butters, Anal. Biochem. 284 (2000) 136, doi:10.1006/abio.2000.4678. [7] K. Oka, M. Yamamoto, T. Nonaka, M. Tomonaga, Neurosurgery 38 (1996) 733. [8] S.C. Churms, J. Chromatogr. A 720 (1996) 75. [9] X. Li, Y. Wang, J. Wang, J.P. Fawcett, L. Zhao, J. Gu, Rapid Commun. Mass Spectrom 21 (2007) 247, doi:10.1002/rcm.2822. [10] V.P. Shah, K.K. Midha, J.W.A. Finlay, H.M. Hill, J.D. Hulse, J.J. McGilveray, G. McKay, K.J. Miller, R.N. Patnaik, M.L. Powell, A. Tonelli, C.T. Viswanathan, A. Jacobi, Pharm. Res. 12 (2000) 1551.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Rapid quantification of miglustat in human plasma and cerebrospinal fluid by liquid chromatography coupled with tandem mass spectrometry Jérôme Guitton a,b,c,∗ , Sylvie Coste d , Nathalie Guffon-Fouilhoux c,e , Sabine Cohen d , Monique Manchon d , Marc Guillaumont d a
Laboratoire de ciblage thérapeutique en cancérologie, Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, F-69495 Pierre Bénite, France Laboratoire de Toxicologie, Université de Lyon 1, ISPB, Faculté de pharmacie, F-69008 Lyon, France Laboratoire Inserm U820 Métabolomique et maladies métaboliques, Faculté de médecine A. Carrel, Université de Lyon, F-69372 Lyon, France d Laboratoire de biochimie-toxicologie, Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, F-69495 Pierre Bénite, France e Service des Maladies Métaboliques, Hospices Civils de Lyon, Hôpital E. Herriot, F-69437 Lyon, France b c
a r t i c l e
i n f o
Article history: Received 24 July 2008 Accepted 26 November 2008 Available online 3 December 2008 Keywords: Miglustat OGT 918 Deoxynojirimycin Mass spectrometry Liquid chromatography
a b s t r a c t Miglustat (OGT 918) is an iminosugar recently introduced in therapeutic as potential alternative therapy in disorders found in several diseases such as Tay-Sachs, Gaucher or Niemann-Pick diseases. A highly sensitive liquid-chromatography–electrospray tandem mass spectrometry (LC–MS/MS) assay was developed for the quantification of miglustat in human plasma and cerebrospinal fluid (CSF). The sample preparation consists in a simple protein precipitation with a mixture of acetonitrile/methanol (75/25) which yields 100% recovery. The isocratic separation utilizes an Atlantis Hilic (3 m, 150 mm × 2.1 mm) column, with a mobile phase of acetonitrile/water/ammonium acetate buffer (75/10/15, v/v/v) delivered at 230 l/min. Selected reaction monitoring (SRM) mode was used with the transitions m/z 220 → 158 for the miglustat and m/z 208 → m/z 146 for the miglitol (internal standard). Good linearity was observed in a range from 125 to 2500 ng/ml and from 50 to 1000 ng/ml, for plasma and CSF, respectively. The within-run precision of the assay was less than 6%, and the between-run run precision was less than 6.5%, for six replicates at each of three concentrations and evaluated on three separated days for both plasma and CSF mediums. Assay accuracy was in the range of 98–106.5%. Stability of miglustat was reported under a variety of storage conditions. The miglustat concentrations in two children are presented to demonstrate the clinical interest of this new method. © 2008 Elsevier B.V. All rights reserved.
1. Introduction N-Butyl-1-deoxynojirimycin (OGT 918) is an iminosugar introduced in therapeutic with the name of miglustat (Zavesca) [1]. Miglustat is a competitive inhibitor of the ceramide-specific glucosyltransferase which catalyzes the first step in the synthesis of glycosphingolipids (GSLs), essential components of the eukaryote cell membranes [2]. Several genetic mutations may produce a pathogenic accumulation of GSLs into lysosomes leading to the GSLs storage disorders found in several diseases such as Tay-Sachs, Gaucher or Niemann-Pick diseases [3]. These diseases are characterized by visceral disorders and by progressive neuropathic malfunctions with important clinical heterogenic manifestations [4]. Unfortunately, until now, treatments were limited to supportive
∗ Corresponding author at: Laboratoire de ciblage thérapeutique en cancérologie, Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, F-69495 Pierre Bénite, France. E-mail address: [email protected] (J. Guitton). 1570-0232/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2008.11.040
care and management of intervening problems [5]. In this condition, miglustat has been recently proposed as a potential alternative therapy. As far as we know, only one assay has been reported for the analysis of N-alkylated iminosugars in biological samples [6]. This method, based on the high-performance cation exchange chromatography coupled to the pulsed amperometric detection, is time-consuming since compound extraction and purification require more than 2 h. Moreover no data in term of methodological validation was provided [6]. In one other study, the use of MALDI-ToF MS technique for the quantification of miglustat is only mentioned, but no analytical description was submitted [4]. The aim of the present study was to develop a simple and rapid assay for the quantification of miglustat from human plasma and cerebrospinal fluid (CSF). Due to the high polarity of miglustat a hydrophilic interaction liquid chromatography (Hilic) was selected, coupled with a quadrupole tandem mass spectrometry to enhance the selectivity of the method.
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Ionization suppression study was performed using a postcolumn infusion of 1 g/ml solution of miglustat and miglitol while a blank of plasma or CSF was injected. 2.4. Sample preparation
Fig. 1. Chemical structures of miglustat (N-butyl-1-deoxynojirimycin – MM: 219) (A) and miglitol (N-hydroxyethyl-1-deoxynojirimycin – MM: 207) (B).
2. Experimental 2.1. Reagents and chemicals The standard of miglustat was from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and was kindly furnished by Dr M-T Vanier (Inserm U820, France). Miglitol was from Pfizer (New York, USA). N-Methyldeoxynojirimycin (N-Methyl-dnj), reagent-grade ammonium acetate, ammonia (30%) were supplied from Sigma (St Quentin Falavier, France). HPLC-grade acetonitrile (ACN) was obtained from Merck (Darmstadt, Germany). Methanol was from Carlo Erba (Milano, Italy). The water used throughout the study was produced from a deionized water system Milli-Q (Millipore corporation, MA, USA). Drug free normal human plasma was provided by the regional blood bank (EFS rhône-Alpes, France). ReconstitutedCSF (R-CSF) preparation derived from a method proposed by Oka et al. and contains: glucose (0.8 g/l), albumin (0.2 g/l), NaCl (7.3 g/l), NaHCO3 (1.9 g/l), the mixture was adjusted at pH 7.4. All the reagents were supplied by Sigma [7]. Stock solutions of miglustat and miglitol were prepared in methanol at a concentration of 1 mg/ml, and stored at +4 ◦ C (see Fig. 1 for the structures). These solutions were then diluted with water to yield a series of spiking standard solutions. 2.2. Chromatographic conditions and instrumentation The chromatographic system used consisted of a Surveyor MS quaternary pump, Surveyor autosampler injector and column thermostater (ThermoElectron, San Jose, USA). HPLC isocratic separation of analytes was performed on a Waters Atlantis Hilic 3 m, 150 mm × 2.1 mm analytical column (Waters, Milford, USA) maintained at 25 ◦ C. The eluant was a mixture of acetonitrile/water/buffer (75/10/15, v/v/v) delivered at 230 l/min. The buffer consisted of ammonium acetate 100 mM, pH 5. 2.3. Mass spectrometric conditions and instrumentation The detector was a Quantum-Ultra (ThermoElectron, San Jose, USA) triple quadrupole mass spectrometer equipped with an IonMax API source. The instrument was operated in positive ion mode with electrospray (ESI) source. The position (x, y, z) of the ESI probe was optimized with miglustat. Argon was used as collision gas at 1.5 mTorr. Spray voltage and capillary temperature were set at 3.5 kV and 300 ◦ C, respectively. The pressures for the nitrogen sheath gas, auxiliary gas and sweep gas were maintained at 30, 10 and 5 units (units refer to an arbitrary value set by the X-calibur software). For the quantification of miglustat and the miglitol (internal standard), the transitions m/z 220 (Q1) → m/z 158 (Q3) (miglustat) and m/z 208 (Q1) → m/z 146 (Q3) (miglitol) were monitored using the selected reaction monitoring (SRM) with 500 ms dwell time per channel. The first (Q1) and the third (Q3) quadrupole were set with full-width at half maximum height of 0.7 Th. Collision energy was set at 24 eV and 22 eV for miglustat and miglitol, respectively.
All samples were prepared by simple protein precipitation. At 10 l of plasma was added 50 l of internal standard (75 g/ml), then 1 ml of mixture acetonitrile/methanol (75/25) was introduced. Samples were vortexed for 30 s, and centrifugation was performed at 13,000 × g for 10 min. For CSF, at 20 l of sample was added 20 l of internal standard (75 g/ml), then the preparation was the same as for plasma. At last, in both cases, 5 l of the supernatant was injected into the HPLC device by an autosampler maintained at +10 ◦ C. 2.5. Calibration curve and validation procedure 2.5.1. Calibration Calibration curves were prepared with blank plasma or R-CSF samples. Miglustat was added to plasma to yield final concentrations of 125, 250, 500, 1250, 2500 ng/ml and to R-CSF to yield final concentrations of 50, 100, 250, 500, 1000 ng/ml. Two clinical studies have shown that the plasma and the CSF concentrations were in these ranges of concentration when children were treated with miglustat for GSLs storage disorders diseases [1,5]. A total of 12 calibration curves in plasma and 5 in CSF were generated during the entire validation process. Peak area ratios of miglustat to the internal standard measured at each nominal concentration were used to construct weighted (1/concentration) least-square linear regression curves. 2.5.2. Precision and accuracy The between-run precision (BRP), the within-run precision (WRP) and accuracy of the method were evaluated on three separate days by six replicates analysis of quality control (QC) containing miglustat at concentration of 150, 750, 1500 ng/ml and 75, 375, 750 ng/ml for plasma and R-CSF, respectively. An estimate of the BRP was obtained by one-way analysis of variance (ANOVA) for each test concentration using “run day” as the classification variable. The WRP was determined as WRP = 100 ×
MSwit /GM . The BRP was estimated as BRP = 100 ×
(MSbet − MSwit )/n /GM . MSwit , MSbet , n, GM, represented
the within-groups mean square, the between-groups mean square, the number of replicate observations within each run and the grand mean, respectively. These parameters were calculated using the software Statview for windows version 5.0 (SAS institute, Cary, USA). The accuracy was expressed as a percentage of bias ((mean value − nominal value)/nominal value × 100). 2.5.3. Recovery, specificity and sensitivity The recovery was conducted at three concentrations (QC) by comparing the mean response of extracted spiked plasma samples, with those obtained from direct injection of the same amount dissolved in the mobile phase. The specificity of the method was tested by visual inspection of chromatograms of extracted human plasma samples from six donors for the presence of interfering peaks. The lower limit of quantification (LLOQ) was defined as the lowest standard concentration which produced assay result within ±20% of the nominal concentration, with a coefficient of variation less than 20%. 2.5.4. Stability Stability studies were conducted, under a variety of conditions, in blank plasma spiked at 150, 750, 1500 ng/ml (QC) in
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Fig. 2. MRM chromatograms for miglustat (m/z 220 → 158) (left) and miglitol (internal standard) (m/z 208 → 146) (right) in blank plasma (a), in R-CSF (b), in plasma spiked with miglustat at 125 ng/ml and miglitol at 3750 ng/ml (c); R-CSF spiked with miglustat at 50 ng/ml and miglitol at 1500 ng/ml (d).
duplicate and in R-CSF spiked at 75, 375, 750 ng/ml (QC) in duplicate. The auto-sampler stability (20 h at +10 ◦ C) of the compounds after extraction from spiked plasma was tested. The stability after storage of plasma and R-CSF aliquots at +4 ◦ C was performed by re-analyzing samples once a day for 3 days. The freeze–thaw stability of miglustat was tested following five and three cycles at −20 ◦ C in plasma and R-CSF, respectively. The samples were brought to room temperature, well vortexed, and extracted immediately. Furthermore, the long-term freezer stability at −20 ◦ C was tested with plasma samples from patients treated with miglustat and with spiked plasma samples and re-analyzed 6 months later the first analysis. At last, the stability of miglustat was checked at room temperature by analyzing samples every hour for 6 h.
2.6. Patient samples Plasma and CSF samples were obtained from two children treated by a daily miglustat dosage over 6 months. These patients were included in a study approved by an ethic committee. Each child and/or their parents gave written informed consent. Blood and CSF samples were collected on day 0 before treatment and 6 months later. Blood samples (5 ml) were collected in lithium heparin glass tubes and tubes were gently inverted several times. Then blood was centrifuged at 3000 × g for 10 min at +4 ◦ C, and plasma was transferred to 5 ml polypropylene tubes and kept at −20 ◦ C until analysis. CSF samples were collected in 5 ml glass tubes, centrifuged at 3000 × g for 10 min at +4 ◦ C and supernatants were kept at −20 ◦ C until analysis.
Fig. 3. Regions of ionization suppression in plasma and in CSF for miglustat (left) and miglitol (right). Chromatograms were obtained after injection of 5 l of blank plasma (a) and CSF (b) after extraction procedure. Experiment was carried out using a post-column infusion of a 1 g ml−1 solution (speed infusion: 5 l min−1 ). Arrow indicates the retention time of compounds.
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3. Results and discussion 3.1. Liquid chromatography During the method development, we spiked with miglustat three blank CSF samples from three patients untreated with miglustat. No difference was observed between these samples and R-CSF spiked at the same concentration. Consequently, we used a surrogate matrix for CSF throughout the assay. Both miglustat and miglitol eluted as sharp peaks and plasma, or CSF, matrix components did not interfere with the analysis (Fig. 2). With the chromatographic conditions described a retention times for miglustat (3.7 min) and miglitol (3.9 min) were found. Consequently, a short run-time of 5 min was achieved. Chromatographic separation performed with Hilic column allows to obtain peak shape, retention and ionization of high polar compounds such as miglustat and miglitol. This mode of chromatography has been used for many years for polar compounds as, for example sugars [8]. Ionization suppression regions were found at 1.0–2.0 min for the plasma for both compounds, and 2.0–2.5 min for the CSF in the case of miglustat (Fig. 3). The retention of miglustat and miglitol was adequate to avoid a modification of the signal. 3.2. Mass spectrometry N-Methyl-dnj was, in the first intention, selected as internal standard. However, this compound exhibited an important variation of response during a run. No explanation and no solution were found as satisfactory and miglitol was tested as internal standard. This compound did not exhibit this signal variation and was selected as internal standard. During the first step, due to the polarity of miglustat and miglitol, the ionization conditions were tuned by infusing a mixture of both products using the electrospray ion source (ESI). However, tests performed with an APCI source also showed a response roughly in the same range as the one obtained with ESI. In both cases, miglustat and miglitol formed predominantly protonated molecules ([M+H]+ ) in the mobile phase used in the present study. During the second step, optimal ionization conditions were set and ESI response was found to be more stable and slightly higher, consequently this source was selected for the rest of the assay. The most sensitive daughter ions for miglustat and miglitol were found at m/z 158 and 146, respectively (Fig. 4). The MS/MS parameters were optimized to maximize the response for miglustat parent/daughter ion of m/z 220 → 158 and for the miglitol of m/z 208 → 146 in the positive ion mode. Li and al. proposed a fragmentation pathway for the miglitol corresponding to the loss of the N-alkyl-chain and a dehydratation on the deoxynojirimycin ring (Fig. 5) [9]. Miglustat and miglitol are structural analogues, so, if this fragmentation pathway is to be retained, the production ion spectra for the two compounds should show similar fragmentation patterns with a predominant daughter ion at m/z 146. However, we observed two different main daughter ions at m/z 158 (miglustat) and m/z 146 (miglitol). For the two compounds, the mass spectra showed ions corresponding to a successive dehydratation (miglustat: 220, 202, 184, 166; miglitol: 208, 190, 172, 154) and a parent-main daughter ion difference equal to m/z 62 (Fig. 4). So, another fragmentation pathway has been proposed based on the opening of the deoxynojirimycin ring and no fragmentation on the N-alkyl-chain. The Fig. 6 presents the fragmentation proposed for miglustat. The fragmentation patters is the same for the miglitol with a mass shift corresponding to the N-alkylchain differences. Only labeled compounds on different positions should allow the thorough determination of the fragmentation patterns.
Fig. 4. Product ion mass spectrum (partial fragmentation) of miglustat (A) and miglitol (B).
3.3. Validation The calibration curves were obtained by weighted 1/[concentration] least-squares linear regression analysis of miglustat concentration versus peak area ratio of miglustat/miglitol area ratios. Standard curves exhibited excellent linearity in the range of 125–2500 ng/ml for plasma, and 50–1000 ng/ml for CSF, with coefficients of correlation greater than 0.996. The resulting assay precision and accuracy data are presented in Tables 1 and 2. Assay accuracy and precision are determined by the validation QC samples. The within-run precision of the assay was less than 6%, and the between-run run precision was less than 6.5%, for each concentration on three QC samples and for both plasma and CSF mediums. Assay accuracy was in the range of 98–106.5%. The lower limit of quantification of miglustat in both mediums was set at the lowest standard concentration. However, in order to evaluate the performance of this assay, plasma samples were also spiked at 25 ng/ml. For this concentration precision was within 20% and accu-
Fig. 5. Previous fragmentation pathway proposed for the miglitol [9].
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Fig. 6. Proposed fragmentation pathways for the miglustat leading to the main ions observed in the mass spectrum.
racy between 80 and 120%. The limit of quantification of the method could be easily set lower if necessary by using a volume sample higher or by reducing the volume of precipitation mixture. As suggested by Shah et al. the high QC should be near the upper boundary of the calibration curve [10]. In our study, this QC is between the upper limit of quantification and the next lowest point and could be set at higher concentration (2000 ng/ml for example). Since previous pharmacokinetic data indicated that upper plasma concentrations are mostly in the range 1000–1700 ng/ml, we choose to set the high QC at 1500 ng/ml. The extraction recovery of miglustat and miglitol was determined by comparing peak areas from plasma samples obtained after single step protein precipitation with those obtained from direct injections of standard solutions. The mean recovery of miglustat was 101, 102 and 102% for low, medium and high QC, respectively (n = 3). The mean recovery of miglitol was 101% (n = 9) which is the concentration used in plasma for the present study.
Table 1 Inter-day validation of the determination of miglustat in plasma and in cerebrospinal fluid (CSF). Concentration (ng/ml) Spiked
Precision (%) (between run)
Accuracy (%)
Found (mean ± S.D.) 10 15 30 51 33
7.8 6.3 5.9 4.2 1.3
100.8 98.1 102.3 98.0 100.8
CSF (n = 5) 50 100 250 500 1000
3.2 7 15 21 21
6.3 7.2 6.2 4.3 2.0
102.9 98.7 100.2 98.2 101.6
± ± ± ± ±
Stock solutions of miglustat and miglitol in methanol were stable for at least 6 months without observable degradation when stored at +4 ◦ C. After extraction from spiked plasma, the stability of miglustat and miglitol was demonstrated for at least 20 h when vials were maintained in the autosampler at +10 ◦ C. Consequently, the present method allows analyzing numerous samples within a single run. In spiked human plasma or R-CSF, miglustat was stable for at least 3 days when samples were stored at +4 ◦ C. In both mediums, miglustat is stable for at least 6 h at room temperature. In frozen human plasma and in R-CSF, miglustat has been found to be stable for 6 months at −20 ◦ C. Finally, no significant degradation (<10%) was observed for miglustat after five or three freeze–thaw cycles in plasma and R-CSF, respectively. This data is in agreement with the observations of Li et al. who showed that miglitol was stable under a variety of conditions [9]. 3.5. Application of the method The suitability of the developed method for clinical use was demonstrated by the determination of miglustat in plasma and CSF Table 2 Assessment of accuracy and precision in plasma and in cerebrospinal fluid (CSF).
Plasma (n = 12) 125 126 ± 250 245 ± 500 512 ± 1250 1224 ± 2500 2519 ± 51.5 99 250 491 1016
3.4. Stability
Data from calibration curves prepared as a single replicate and analyzed on different days.
Concentration (ng/ml)
Precision (%)
Accuracy (%)
Spiked
Found (mean ± S.D.)
Within-run
Between-run
Plasma 150 750 1500
160 ± 9 771 ± 43 1505 ± 92
5.5 3.4 2.9
1.7 5.3 6.4
106.5 102.9 100.4
CSF 75 375 750
75.7 ± 5.8 375 ± 17 735 ± 35
5.8 3.0 4.2
6.1 4.0 2.7
101.0 100.2 98.0
Data from six replicates for each concentration and analyzed on three different days.
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Fig. 7. MRM chromatograms for miglustat (m/z 220 → 158) (left) and miglitol (internal standard) (m/z 208 → 146) (right) in plasma from child before treatment (a); in CSF from child before treatment (b); and response in plasma (miglustat 1679 ng/ml) (c) and in CSF (miglustat 243 ng/ml) (d) for child treated over 6 months. Miglitol was present at 3750 ng/ml in plasma (a and c) and at 1500 ng/ml in CSF (b and d).
samples from two children treated by miglustat. The concentrations determined with both mediums were in agreement with previously published values [1,5]. No endogenous peaks were found interfering with miglustat or miglitol (Fig. 7). 4. Conclusion The new LC–MS/MS method described here provides major advantages in terms of simplicity of sample extraction, selectivity of detection and turnaround time as compared with previous HPLC method [6]. This method has been successfully applied to analyze miglustat concentrations in human plasma and CSF. This assay meets the requirements of bioanalytical methodology validation, providing good accuracy and precision. The present method permits the analysis of plasma and CSF human samples in a range of concentrations which are sufficiently sensitive to allow monitoring after oral administration of miglustat in GSLs diseases.
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