Methodology
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Application of a high-throughput, parallel HPLC system for quantitative chiral analysis of pantoprazole
Background: Chromatographic separation of enantiomers is considered a task in analytical chemistry particularly for high sample throughput. This paper describes a high-throughput parallel HPLC–MS/MS method for the determination of pantoprazole enantiomers. Results: Baseline separation of pantoprazole enantiomers was achieved on a Chiralcel OZ-RH column in a run time of 4.5 min. Assays for enantiomers were linear with satisfactory intra- and inter-day precision and accuracy. The assay was suitable for high-throughput analysis as shown by its successful application to a chiral PK study in beagle dog. Conclusion: A high-throughput parallel HPLC–MS/MS assay for pantoprazole has been developed and validated. This method provides nearly twofold increased sample throughput, and was shown to be suitable for application in PK studies. First draft submitted: 16 June 2015; Accepted for publication: 5 October 2015; Published online: 30 November 2015
Pantoprazole is a benzimidazole derivative used clinically as an antiulcerative agent in the treatment of reflux esophagitis, Zollinger–Ellison syndrome and other acidrelated hypersecretory gastrointestinal disorders [1–3] . It belongs to the class of drugs called proton pump inhibitors which act as selective and irreversible inhibitors of proton-pump (H +/K+ -ATPase) activity in the parietal cells of the stomach [4,5] . Pantoprazole is a chiral drug due to the presence of an asymmetrically substituted sulfur atom that reportedly undergoes stereoselective metabolism by the hepatic CYP450 system [6–9] . Clinical studies have shown that (-)-pantoprazole is more active than its antipode in the management of gastroesophageal reflux disease and esophagitis symptoms [6] . Chromatographic separation of enantiomers is considered a challenging task in analytical chemistry. LC–MS/MS has been applied to the chiral analysis of pantoprazole by Barreiro et al. who developed a column-switching method [10] , but the chromatographic run time was 40 min and
10.4155/bio.15.215 © 2015 Future Science Ltd
peak width was over 5 min. Chen et al. also established a chiral LC–MS/MS method for pantoprazole but again the run time was long at 10 min [11] . Clearly these methods are not applicable to high-throughput chiral PK studies which can potentially involve hundreds of samples. Over recent years considerable effort has been devoted to increasing the throughput of LC–MS analysis. One strategy is to use parallel-column LC–MS systems which process samples on different columns using a staggered technique [12–19] . Klavins et al. [15] established a parallel LC column method that performed orthogonal hydrophilic interaction chromatography and reversedphase chromatography within the same analytical run. Zhao et al. [17] developed a metal-free multiple-column nano-LC technique that increased the sensitivity and throughput of phosphopeptide analysis. In proteomic analyses, parallel column LC systems are being increasingly applied to increase sample throughput [18,19] . Some studies have used a multiplexing parallel
Bioanalysis (2015) 7(23), 2981–2990
Hao Wang‡,1, Yantong Sun‡,2, Yan Yang‡,1, Wei Guo3, Mi Zheng1, Weiping Zhang3, J Paul Fawcett4 & Jingkai Gu*,3 College of Life Sciences, Jilin University, Changchun 130012, PR China 2 School of Pharmaceutical Sciences, Jilin University, Changchun 130012, PR China 3 Research Center for Drug Metabolism, Jilin University, Changchun 130012, PR China 4 School of Pharmacy, University of Otago, PO Box 56, Dunedin, New Zealand *Author for correspondence: Tel.: +86 431 8515 5381 Fax: +86 431 8515 5380
[email protected] ‡ Authors contributed equally 1
part of
ISSN 1757-6180
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Methodology Wang, Sun, Yang et al.
Key terms Pantoprazole: A proton pump inhibitor drug that inhibits gastric acid secretion, which is used for short-term treatment of erosion and ulceration of the esophagus caused by gastroesophageal reflux disease. Parallel-column LC–MS system: To increase the throughput by multiplexing two or more LC systems into a single MS detector. MPX TM-2 system: A parallel LC–MS/MS system could increase throughput by multiplexing two HPLC systems into a single mass spectrometer. High-throughput analysis: Allow researchers to analyze more samples in a shorter time period.
technique as exemplified by the commercially available MUX interface [12] , which consists of four or eight electrospray ionization (ESI) emitters that can sequentially inlet samples using a rotating aperture. In this paper we report the application of the new MPX TM -2 system (AB Sciex) to the chiral analysis of pantoprazole. It multiplexes two HPLC systems into a single mass spectrometer, which provides nearly twofold increased throughput compared with a generic single column system. Timing of injection, valve switching, start of separation and MS data collection are all controlled by a single software package. The system allows the use of a variety of settings, such as columns, mobile phases, acquisition windows, ion source and MS conditions that enable its wide applications. In this case the system was applied to the development and application of a high-throughput analysis for pantoprazole. Experimental
system consisted of one loading pump (LC-20ADXR) and two HPLC instruments (Shimadzu, Kyoto, Japan) each of which included two LC-20ADXR pumps and an SIL-20AXR autosampler but sharing a DGU-20A3 degasser and CTO-20AC column oven. The instruments were combined with two MXT715-000 6-position selector valves (IDEX Health & Science, USA) and one FCV-20AH2 valve (Shimadzu, Kyoto, Japan) (Figure 1) . The MPX TM-2 system was coupled to a Qtrap 5500 mass spectrometer (AB Sciex, Ontario, Canada) equipped with an ESI source operating in the positive ion mode. Chromatographic conditions
Chromatographic separation was carried out on a Chiralcel OZ-RH column (150 × 4.6 mm, 5 μm) (Daicel Corporation, Osaka, Japan) which incorporates a stationary phase of cellulose tris (3-chloro-4-methylphenylcarbamate) coated on a silica support. The column temperature was maintained at 25°C. The mobile phase was acetonitrile: 0.1% formic acid (40:60, v/v) delivered at a flow rate of 0.8 ml/min. The column effluent was split to deliver 400 μl/min into the detector by means of a T-piece. The flow rate of the loading pump was 0.1 ml/min. The MPXTM-2 system consisted of two HPLC streams (stream 1, stream 2) and one loading stream. The function of the loading stream was to transfer samples from autosampler to column using a slow flow rate (0.1 ml/min) over a short time (5 s). The two autosamplers injected samples alternately every 4.5 min. While stream 1 was being separated on the chiral column, stream 2 was being detected in the mass spectrometer so that the runtime of each sample was shortened to 4.5 min.
Chemicals & reagents
(±)-Pantoprazole sodium (>99.0% purity) was obtained from the Hunan Warrant Pharmaceutical Co., Ltd (Hunan, PR China). (+)-pantoprazole sodium, (-)-pantoprazole sodium (>99.0% purity) and (-)-lansoprazole (>99.0% purity) for use as internal standard (IS) were obtained from Shenyang Pharmaceutical University (Shenyang, P R China). HPLC-grade acetonitrile were purchased from Sigma–Aldrich (MO, USA). Formic acid was purchased from Beijing Chemical Plant (Beijing, P R China). All other chemicals were commercially available analytical grade materials. HPLC-grade water was prepared using the Millipore system (Millipore, MA, USA). Instrumentation
An MPXTM-2 system equipped with an Analyst® 1.5.1 workstation (AB Sciex, Ontario, Canada) was employed for the determination of pantoprazole enantiomers. The
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MPX TM-2 system & mass spectrometer conditions
MS settings were optimized by introducing solutions into the ESI source via a syringe pump. The curtain, nebulizer and turbo gases were nitrogen set at 30, 30 and 50 psi, respectively. The source temperature was 550°C, and ion spray voltage 5500 V. Analytes and IS were detected using multiple reaction monitoring (MRM) at unit resolution of the transitions of the protonated molecular ions of pantoprazole at m/z 384.1→200.0 and IS at m/z 370.1→252.0 with collision energies of 16 eV and 14eV, respectively, and declustering potentials both of 40 V. Data acquisition and integration were performed by Analyst software (version 1.5.2) and MPXTM-2 Driver software. Preparation of calibration standards & QC samples
Stock solutions of (+)- and (-)-pantoprazole were prepared in acetonitrile-water (90:10, v/v) and diluted
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Application of a high-throughput, parallel HPLC system for quantitative chiral analysis of pantoprazole
Methodology
To waste
2 3
1 6
A 4
5
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Column 1
Position 1 Pump A1/B1
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1 C 6 4 5
Mass spectrometer
Position 1 2 3
1 6
B 4
Autosampler 2
Column 2
5 Stream 1
Position 2
Loading pump
Stream 2 Loading stream
Figure 1. Schematic diagram of the instrumental setup for the MPX TM-2 system.
with the same solvent to obtain a mixture containing 500 ng/ml of each enantiomer. Calibration standards were prepared through serial dilution of the mixture with blank dog plasma to give concentrations of 0.5, 1.5, 5, 15, 50, 150 and 500 ng/ml for each enantiomer. QC samples were prepared independently in the same way at concentrations of 1.5, 15 and 400 ng/ml of each enantiomer. A 1.0 mg/ml stock solution of IS in acetonitrile-water (90:10, v/v), was also diluted with the same solvent to give a 500 ng/ml IS working solution. All solutions were stored at 4°C, and calibration standards and QC samples were stored at -80°C and brought to room temperature before use. Sample preparation
Frozen plasma samples were allowed to thaw at room temperature and vortex-mixed prior to analysis. An aliquots (100 μl) of IS working solution was added to 100 μl plasma, after which 600 μl cold acetonitrile was added and samples vortex-mixed for 45 s. Precipitated protein was removed by centrifugation at 4°C, 17,000 × g for 5 min after which 10 μl supernatant was injected into the LC–MS system. Assay validation
The method was validated according to the guidelines of the US FDA [20] . Selectivity of the method was assessed by analysis of six individual lots of dog blank plasma spiked at the lower
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limit of quantitation (LLOQ). Linearity was evaluated by preparation of calibration curves in duplicate on three separate batches and analyzing by weighted (1/x2) least squares linear regression based on peak area ratios of analyte to IS. Intra- and inter-day precision and accuracy were evaluated by assay of six replicates of QC samples prepared from three separate batches and considered acceptable if precision ≤15% and accuracy ± 15%, except at LLOQ where ≤20% was acceptable. Matrix effects were assessed by comparing the peak areas of samples spiked after preparation with peak areas of corresponding standard solutions prepared in water. Recovery was determined by comparing peak areas of samples spiked before and after sample preparation. Matrix effects and recovery determinations involved assay of four replicates of QC samples, which should be consistent and precise. In addition to chemical stability, chiral inversion of pantoprazole enantiomers should be considered during sample storage and preparation, especially for sulphoxide drugs [11] . Stability was evaluated by analysis of QC samples of (+)-pantoprazole, (-)-pantoprazole and (±)-pantoprazole in triplicate under the following conditions: at room temperature for 4 h, after storage at -80°C for 1 month and after three freeze-thaw cycles. Stability of processed samples was also evaluated after storage at 4°C for 24 h. Accuracy of each enantiomer should be within 15% for the stability evaluation. To evaluate the feasibility of the method for analyzing plasma samples at concentrations >500 ng/ml, dilu-
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Methodology Wang, Sun, Yang et al.
Max. 6.7e4cps. L-PAN
100
6.82 R-PAN
80 Rel. int. (%)
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Figure 2. Schematic description of the steps performed on one analytical column.
tion integrity was performed by analyzing six replicates of plasma samples with concentrations of 1200 ng/ml after threefold dilution with blank dog plasma. Applications to PK studies
The validated method was applied to a PK study of pantoprazole sodium after a 10 min continuous intravenous infusion of 0.5 mg/kg of (+)-pantoprazole sodium, or 0.5 mg/kg (-)-pantoprazole sodium or 1.0 mg/kg (±)-pantoprazole sodium to six beagle dogs. Venous blood samples were collected into heparinized tubes before the dose and at 1, 5, 10 (end of infusion), 15, 20, 30, 45 min, 1, 1.5, 2, 3 and 4 h after initia-
tion of the infusion. Plasma samples were collected immediately by centrifugation at 15,000 × g for 5 min and stored at -80°C until analysis. All procedures were approved by the Animal Ethics Committee of Jilin University. Results & discussion Method development Method development for the MS/MS detection
In this assay, pantoprazole enantiomers and IS showed strong and stable signals using ESI in the positive ion mode. The [M+H]+ ions at m/z 384.1 for pantoprazole and m/z 370.3 for the IS (lansoprazole) gave 1.48 2.08
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Initial
rea m
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St
100
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50 Acquisition 0
0
4.5
Acquisition 9.0
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Stream 1
18.0
Time (min) Figure 3. Theoretical representation of the cycles involving two streams controlled by the MPX TM-2 system. The acquisition window is the time when the mass spectrometer acquires and integrates the data.
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Application of a high-throughput, parallel HPLC system for quantitative chiral analysis of pantoprazole
Methodology
Relative retention time (min)
3.0 2.5 2.0 1.5 1.0
(-)-pantoprazole (+)-pantoprazole IS
0.5
0 Column # Batch
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2
Val 01
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Ana 01
1
2
Ana 02
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Ana 03
1
2
Ana 04
1
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Ana 05
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Figure 4. Relative retention times of (+)-pantoprazole, (-)-pantoprazole and IS during the assay. For validation batches (Val), n = 9 for each point; for the actual batches (Ana), n = 3 for each point.
major fragment ions at m/z 200.0 and m/z 252.0, respectively, in the product ion spectra. These transitions were selected for MRM as they were free from interference and showed a stable response. HPLC method development on a single column
When developing a parallel column method using MPX-2 system, the theoretically efficiency of the method is equal to analysis time/acquisition window time. The maximum increase in efficiency of the MPX-2 system is twofold. A shorter analysis time with a shorter acquisition window can increase sample throughput significantly when developing a parallel column method using MPX-2 system. In this assay, a Chiralcel OZ-RH column was chosen for determination of pantoprazole enantiomers because of its satisfactory chromatographic behavior and enantioselective resolution. Acetonitrile as organic modifier gave better resolution of enantiomers than methanol and the inclusion of formic acid improved the MS response. The optimum mobile phase composition in terms of enantioselectivity, peak shape and response was acetonitrile: 0.1% formic acid water (40: 60, v/v). Baseline separation (R = 1.7) of pantoprazole enantiomers was achieved within 9.0 min using a flow rate of 0.8 ml/min. MPX TM-2 method development
The configuration of the MPXTM-2 system is illustrated in Figure 1 where valve A and B are used for loading samples onto the columns, and valve C is used to select which of the two streams enters the MS. As shown in Figure 2, each sample separation involves a load step consisting of washing the sample injection syringe, injecting the sample into the loop and loading the sample onto the column. Differences in injection volume,
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rinsing speed, sampling speed and rinse mode (with or without needle washing) can affect the duration of the load step. Because the autosampler alone cannot accurately control this duration, the MPXTM-2 system uses a loading pump to load the sample onto the head of the column, and two solvents select valves to select which mobile phase enters the column. The analysis then consists of three regions: the dead time, the no peak region and the peak region. In the analysis of chiral drugs, the length of the traditionally long no peak region is considerably shortened using a parallel HPLC system, such as the MPX TM-2. While the dead time and no peak regions are performed on one separation stream, the MS acquires the data from the other stream. A schematic diagram of the analytical cycle controlled by the MPX TM-2 system is illustrated in Figure 3. At the beginning of a run, stream 1 loads the 1st sample and initiates the separation. After running this sample for 4.5 min, stream 2 initiates a run on column 2 by switching valve B to position 1 (the 2nd sample having been already loaded onto column 2 by the loading pump) and MS begins to acquire data from stream 1. At 8.5 min, data acquisition of stream 1 by the MS is completed and, at 8.5–9.0 min, a 3rd sample is loaded onto the column 1. At 9.0 min, the MS begins to acquire data for the 2nd sample and stream 1 initiates the separation of the 3rd sample. In this way, the MPX TM-2 system provides a maximizing twofold increase in sample throughput. All the above procedures were controlled automatically by the MPX TM-2 Driver software. Reproducibility
To accurately control the retention time for the two streams, the path of the two streams and the separation capability of the two columns should be equal. Chro-
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2985
Methodology Wang, Sun, Yang et al.
Key term Method validation: Validation could assure the precision, accuracy and reproducibility of a bioanalytical method.
matographic stability of the two streams was investigated using retention time and response of the IS based on 90 QC samples (45 injections per column). The results show that the variability of the IS response was less than 15%, and retention time < 0.1 min between the two streams (Figure 4) . Comparison with previous methods
The MPX TM-2 system consists of two fully independent HPLC systems tandem one mass spectrometry, samples from different HPLC system are introducing into the ion source sequentially via the selection valve C (Figure 1) . Time for samples’ injection, analyzing start on each system is completely controlled by the MPX software. Because the two HPLC systems are fully independent, the MPX TM-2 system has a better adaptability. There are three different ways to run the samples: each of two HPLC systems running the same method, two HPLC systems running two different methods and run two sequential sample batches from two HPLC systems. Compared
with previous methods [12–19] , this system is simple for users, since it does not need special emitters, such as MUX [12,18] , or other specially designed requirements [17,19] . Klavins et al. [15] developed a parallel hydrophilic interaction chromatography and RPLC chromatography method, but the two LC systems are not fully independent. The method is used to analyze one sample on the two columns simultaneously, which could not run the same method on each of two columns. The MPX TM-2 system is most likely the Aria LX4 system developed by Cohesive Technologies [21] . The Aria LX4 system is a four column parallel system, which may provide a higher throughput, but has a more complex configuration. Method validation
Selectivity was assessed by comparing the chromatograms of six individual blank plasma samples with the corresponding spiked plasma. Figure 5 shows typical chromatograms of a blank plasma sample, a blank plasma sample spiked at the LLOQ and a plasma sample obtained 1 h after intravenous infusion of pantoprazole sodium. The assay was free of interference and linear over the concentration range 0.5–500 ng/ml with typical linear regression equa-
Max. 25.0 cps. 100 I
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Figure 5. Representative MRM chromatograms for (I) (-)-pantoprazole, (II) (+)-pantoprazole and (III) IS in dog plasma: (A) a blank plasma sample; (B) a blank plasma sample spiked with pantoprazole (0.5 ng/ml); (C) a plasma sample from a dog 1 h after intravenous infusion of 1.0 mg/kg (±)-pantoprazole sodium; (D) IS.
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Bioanalysis (2015) 7(23)
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Application of a high-throughput, parallel HPLC system for quantitative chiral analysis of pantoprazole
Methodology
Table 1. Accuracy and precision for determination of (+)-pantoprazole and (-)-pantoprazole in beagle dog plasma (data are based on three different days, six replicates per day). Analyte
Nominal
Concentration (ng/ml) Observed (mean ± SD)
Intra-day
RSD (%) Inter-day
RE (%)
(+)-pantoprazole
0.50 (LLOQ)
0.50 ± 0.04
8.74
9.13
-0.33
1.50
1.55 ± 0.10
6.74
2.59
3.00
15.0
15.0 ± 0.93
4.13
14.22
-0.02
400
414 ± 5.9
0.79
1.50
3.39
(-)-pantoprazole
0.50 (LLOQ)
0.46 ± 0.03
7.28
1.83
-9.49
1.50
1.54 ± 0.11
5.71
12.57
2.33
15.0
14.7 ± 0.79
5.08
7.25
-1.80
400
403 ± 10.2
3.10
2.44
0.69
tions of y = 0.0316x + 0.0019 (r = 0.9970) for (+)-pantoprazole and y = 0.0327x + 0.0023 (r = 0.9968) for (-)-pantoprazole. The LLOQ was 0.5 ng/ml for both pantoprazole enantiomers at a S/N ratio of 14.4 for (+)-pantoprazole, and 17.1 for (-)-pantoprazole. Precision and accuracy were respectively
