Automated simultaneous quantification of the immunosuppressants 40-O-(2-hydroxyethyl)rapamycin and cyclosporine in blood with electrospray-mass spectrometric detection

¹ Medizinische Hochschule Hannover, Institut für Allgemeine Pharmakologie, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.

² Universität Hannover, Institut für Anorganische Chemie, Lehrgebiet Analytische Chemie, D-30167 Hannover, Germany.
^a Author for correspondence. Fax 49 511 532 2794; e-mail Vidal. Christian{at}mh-hannover.de.

	Abstract

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A new analytical method to quantify 40-O-(2-hydroxyethyl)rapamycin (SDZ RAD) and cyclosporine (Cs) simultaneously in blood is presented. The combination of an on-line solid-phase extraction step with an HPLC system coupled to an electrospray mass spectrometer gave excellent specificity, sensitivity, and reproducibility. Aliquots of deproteinized blood samples were injected into the HPLC system and extracted on-line, using a conventional C₁₈ guard column. The extract was eluted from the guard column in the backflush mode and injected into the liquid chromatography–mass spectrometry system. The calibration functions for SDZ RAD and Cs extracted from blood with added analyte were linear from 0.15 to 30 µg/L (r² = 0.999) and from 1.5 to 1000 µg/L (r² = 0.999), respectively. The CVs of peak areas were 6.2% at 10 µg/L SDZ RAD (n = 6) and 6.2% at 100 µg/L Cs (n = 6). Recovery ranged from 84.3% to 102.3% for SDZ RAD and from 81.7% to 92.2% for Cs. The lower limit of detection for both drugs was 0.05 µg/L. A rate of four samples per hour was maintained during the consecutive analysis of SDZ RAD and Cs in >500 blood samples with one single extraction and analytical column. The method described is a powerful tool for the simultaneous determination of SDZ RAD and Cs in blood. It works without time-consuming sample preparation steps and with excellent reproducibility. Because of the detection performance of electrospray mass spectrometry, this system offers flexibility in the working range, which is essential for therapeutic drug monitoring under different conditions.

	Introduction

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Tailored immunosuppressive therapy after organ transplantation continues to be a great challenge. The cyclic peptide cyclosporine (Cs)¹ has been in use as an immunosuppressant for many years. Because Cs can produce untoward reactions, such as nephrotoxcity (1)(2)(3) or neurotoxicity (4) and hypertension (5), the search for alternatives is the object of intensive research activities. With the introduction of tacrolimus (FK-506; Fujisawa, Osaka, Japan) and sirolimus (rapamycin; Wyeth-Ayerst, Princeton, NJ), a generation of highly potent macrolide immunosuppressants has entered the market or is under development. However, administration of both drugs is associated with severe side effects (6)(7)(8). 40-O- (2-hydroxyethyl)rapamycin (SDZ RAD; Novartis Pharma; Fig. 1 ) is currently under clinical investigation in combination with Cs. This is a new therapeutic approach that could lead to new insights into immunosuppressive therapy. Both SDZ RAD and Cs require therapeutic drug monitoring, which is an analytical challenge because the working ranges of both drugs differ by one order of magnitude in concentration.

View larger version (11K): [in this window] [in a new window]   Figure 1. Structure of SDZ RAD (40-O-(2-hydroxyethyl)rapamycin; C53H83NO14). The exact mass is 957.58 atomic mass units; the molecular weight is 958.22.

No analytical tools have been described to date to determine the concentration of SDZ RAD in biological matrices. Like sirolimus, the structure of SDZ RAD is based on a 31-member macrolide lactone. The molecular formula is C₅₃H₈₃NO₁₄, giving a molecular weight of 958.22. Because SDZ RAD, like sirolimus, lacks a sufficient chromophore for sensitive detection in the ultraviolet region, determination by HPLC-UV is of insufficient sensitivity. The need to determine concentrations as low as <1 µg/L requires a detection method of higher sensitivity (9). For Cs, numerous analytical techniques have been developed that generally meet the requirements for routine monitoring. Most of the methods suggested are based on HPLC with ultraviolet detection, which is adequate for the routine determination of Cs concentrations of clinical relevance (typically between 50 and 300 µg/L) (10)(11)(12)(13).

No method has been published that allows simultaneous determination of SDZ RAD and Cs in the range of therapeutic relevance. The aim of our work was to provide an analytical method that offers excellent specificity, sensitivity, and reproducibility, as well as offering maximum flexibility in varying the working range, which is essential for therapeutic drug monitoring under different conditions. In addition to these requirements, when large sample numbers must be measured, a process is needed that allows high sample turnover. At the same time, cost-effectiveness is important. Because pharmacodynamic monitoring for routine use is not available and radioimmunological methods have their limitations, chemical analysis of the drugs in question is currently the method of choice for meeting the requirements of specificity, sensitivity, and reproducibility.

All techniques for chemical analysis of immunosuppressants published thus far require manual or at least off-line sample preparation (9)(10)(11)(12)(14)(15). This is no longer acceptable for large sample turnover from the point of view of reproducibility and the costs for personnel. Therefore, we developed an on-line sample preparation procedure based on a column switching technique and that uses a conventional C₁₈ guard column for simultaneous sample clean-up and enrichment. Some reports dealing with automated sample preparation clearly demonstrated the potential of this technique for the determination of drugs in clinical applications (16)(17)(18)(19). In recent years, various column-switching techniques have been proposed for the determination of a variety of analytes, including some macrolide compounds (20).

This research led to the development of an automated simultaneous determination method for SDZ RAD and Cs in blood that is based on a liquid chromatography–mass spectrometry (LC-MS) system with electrospray ionization (ESI). Here we present data on recovery, sensitivity, linearity, and reproducibility of an on-line solid-phase extraction-LC-ESI-MS system.

	Materials and Methods

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chemicals
Acetonitrile, methanol, and water were of reagent grade and provided by J.T. Baker (Deventer, The Netherlands). Zinc sulfate was obtained from Sigma-Aldrich (Deisenhofen, Germany). The eluent for chromatographic separation was premixed to minimize the gas content and was additionally degassed directly before use in an ultrasonic bath for 15 min. SDZ RAD and Cs as reference substance were kindly provided by Novartis Pharma, Basel, Switzerland. Physiological sodium chloride solution (9 g/L) was obtained from B. Braun Melsungen, Melsungen, Germany. Nitrogen (99.9999%) for the MS system was obtained from Linde AG, Höllriegelskreuth, Germany.

sample preparation and lc separation
On-line sample preparation and chromatographic separation were performed with a system consisting of a Hewlett-Packard 1090 series II liquid chromatograph (Hewlett–Packard) with a slightly modified autoinjector, a Rheodyne 7010 six-port high-pressure switching valve with a two-position Rheodyne 5701 pneumatic actuator and a solenoid valve (Rheodyne, Cotati, CA) remote-controlled by the HP ChemStation software (vide infra) through an external event contact of the HP 1090, and a conventional 10 x 2 mm guard column filled with Nucleosil 100 C₁₈, particle size, 10 µm (Schambeck SFD, Bad Honnef, Germany). For sample enrichment on the guard column, an additional HPLC pump (WellChrom MicroStar K-100, Knauer GmbH) was used. By means of two squeezing valves remote-controlled by the HP ChemStation software through an external event contact of the HP 1090, solvent delivery onto the extraction column could be switched from water (loading, washing) to methanol (reconditioning).

For large volume injection, the autosampler of the HP 1090 was modified as follows: we built in a 500-µL syringe (Hamilton, Bonaduz, Switzerland) instead of the 25-µL conventional model and changed the capillary connection between injection valve and syringe provided by Hewlett-Packard for a capillary with a wider inner diameter (0.26 mm) to guarantee complete suction of the samples. The conventional 25-µL sample loop was exchanged for a 500-µL PEEK® model manufactured by Rheodyne. The guard column used for on-line solid-phase extraction was arranged with capillary connections between ports 1 and 4 of the additional six-port switching valve.

Blood samples (1 mL) were deproteinized by addition of 1 mL of a mixture of 800 mL of methanol and 200 mL of aqueous zinc sulfate solution (0.4 mol/L). After samples were vortex-mixed for 15 s, they were centrifuged for 5 min at 4300g. The supernatant was pipetted into 1.8-mL autosampler vials, and aliquots of 400 µL were directly injected into the HPLC system and extracted on the guard column.

The analytical process consisted of three subsequent phases (Fig. 2 ). In the first step, with the switching valve in position A, samples were injected into the HPLC system and transported through the extraction column by water supplied by the additional HPLC pump at a flow of 0.35 mL/min for 4 min. In the second step, the switching valve was turned pneumatically into position B, allowing a flow of 0.2 mL/min of a mixture of 900 mL of methanol and 100 mL of water provided by the solvent delivery system of the HP 1090 to elute the compounds from the extraction column in the backflush mode. The extract was directly transferred onto the analytical column for separation. After 4 min, the switching valve turned back into position A. Separation of the analytes was achieved on a 250 x 2 mm Hypersil ODS column (particle size, 5 µm) equipped with a 10 x 2 mm guard column filled with identical material (Schambeck SFD, Bad Honnef, Germany) under the same isocratic conditions as for elution from the extraction column, using the solvent delivery system of the HP 1090. Column temperature was 35 °C. In parallel with separation of the compounds on the analytical column, the extraction column was reconditioned with methanol and water, each at a flow of 0.35 mL/min lasting 3 min, provided by the additional HPLC pump.

View larger version (46K): [in this window] [in a new window]   Figure 2. On-line solid-phase extraction. Three subsequent phases involve: (1) loading the precolumn by injecting the sample, then washing with water; (2) elution in the backflush mode; and (3) reconditioning the precolumn with methanol and water, with simultaneous chromatographic separation on the analytical column.

ms
Mass spectrometric measurements were performed on a system manufactured by Hewlett–Packard, consisting of an HP 5989 B MS-Engine mass spectrometer equipped with an API-Electrospray cabinet, HP 59987 A.

After separation of the analytes on the analytical column, as described, the compounds were injected into the electrospray mass spectrometer with pneumatic assistance from a flow of nitrogen at 540 kPa. Nitrogen as the drying gas was supplied at a flow of 10 L/min and a temperature of 350 °C. All mass spectrometric measurements were made in the positive ion mode; the voltage of the capillary was adjusted to -4000 V, the end plate to -3500 V, and the cylinder electrode to -6000 V. The compounds were detected in the selected ion monitoring mode: The mass analyzer was focused on the sodium adduct ions (MNa) of SDZ RAD (m/z 980.6) and Cs (m/z 1223.8), with a dwell time of 0.5 s for each mass. The capillary exit voltage was tuned in a mass-dependent manner to achieve maximum sensitivity for each compound. At m/z 980.6 (SDZ RAD), the voltage was 280 V; at m/z 1223.8 (Cs), it was 300 V. The multiplier was run at 1890 V.

Data acquisition and device control over the HPLC and MS instruments were achieved with the HP LC-MS ChemStation, consisting of the HP G1034C MS ChemStation and the G1047A LC-MS software run on an IBM-compatible PC with an HP-IB interface.

Data were evaluated using the ChemStation Integrator of the HP G1034C MS ChemStation by automated integration of the chromatograms split into the two acquired ion traces corresponding to m/z 980.6 and 1223.8.

calibration and validation
For calibration of the analytical system, samples were prepared by supplementing pooled blood collected from healthy volunteers with appropriate amounts of SDZ RAD and Cs dissolved in acetonitrile. The analytical system was calibrated according to international rules (21), with overlapping series of 10 calibration samples equidistant in concentration within a series. The resulting data points were 0, 0.15, 0.3, 0.45, 0.6, 0.75, 0.9, 1.05, 1.2, 1.35, 1.5, 3, 4.5, 6, 7.5, 9, 10.5, 12, 13.5, 15, 18, 21, 24, 27, and 30 µg/L for SDZ RAD. According to clinical dosing, the concentrations of Cs that were added were 10-fold higher and extended to 1000 µg/L (0, 1.5, 3, 4.5, 6, 7.5, 9, 10.5, 12, 13.5, 15, 30, 45, 60, 75, 90, 100, 105, 120, 135, 150, 180, 200, 210, 240, 270, 300, 400, 500, 600, 700, 800, 900, and 1000 µg/L). For each calibration point, SDZ RAD and Cs were added to three 1-mL samples of blood; the blood samples were deproteinized and prepared for injection. All samples were measured in duplicate.

Validation data consisted of studies of reproducibility and variation through multiple injections and examination of the recovery of the compounds in question from supplemented blood. Intraassay variation was determined by measurements of pooled blood collected from healthy volunteers, supplemented with SDZ RAD and Cs. Six 1-mL samples of blood were prepared, each containing low (10 µg/L Cs, 1 µg/L SDZ RAD), medium (60 µg/L Cs, 6 µg/L SDZ RAD), or high (300 µg/L Cs, 30 µg/L SDZ RAD) drug concentrations; these samples were measured in sequence. For the evaluation of interassay variation, six 1-mL samples of blood, each containing a low, medium, or high (see above) drug concentration, were measured on 6 subsequent days. Drug recovery at low, medium, and high concentrations (see above) was determined in six individually supplemented blood samples measured in sequence with reference to samples prepared with physiological sodium chloride solution instead of blood. Variation of retention times was determined in 24 samples measured in sequence during calibration.

Stability experiments were carried out in such a way that SDZ RAD samples in acetonitrile as well as in blood and in precipitates from blood were measured repeatedly. During storage of SDZ RAD samples in acetonitrile at 25 °C and 4 °C over a 1-week period no loss or change in the material could be determined. The experiments showed that blood and precipitates containing SDZ RAD may be stored for 1 week at 4 °C without any change in the material. An interval up to 4 days at ambient temperature, e.g., in the autosampler during measurement, did not affect sample composition.

	Results

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Recovery data are summarized in Table 1 . The recovery averaged 94.4% for SDZ RAD and 90.2% for Cs. Both SDZ RAD and Cs were detected as the sodium adduct ions (MNa). For both drugs, the signal of the sodium adduct ion was more than 20-fold greater than that of the protonated form (MH). Capillary exit voltage in the electrospray interface was optimal for SDZ RAD at 280 V and for Cs at 300 V and gave the highest intensity without inducing collision-activated fragmentation.

The use of an analytical column with a 2-mm inner diameter reduced flow rates to 0.2 mL/min and increased signal intensity substantially. In addition, it economized solvent consumption. Clean baseline-resolved chromatograms of both compounds are shown in Fig. 3 . Retention times (t_R) were 5.02 min for SDZ RAD and 6.39 min for Cs. Retention times varied by 0.75% for SDZ RAD and 0.96% for Cs (Table 2 ).

View larger version (18K): [in this window] [in a new window]   Figure 3. HPLC-ESI-MS chromatograms of the sodium adduct ions of SDZ RAD (0.6 µg/L) and Cs (6 µg/L) added to blood: (a) total ion current; (b) ion trace of m/z 980.6; and (c) ion trace of m/z 1223.8. The change in baseline appearing around 3 min is attributed to switching of the valve and does not represent any material related to SDZ RAD.

In the working ranges of 0.15–30 µg/L SDZ RAD and 1.5–1000 µg/L Cs, the calibration curves in blood were linear, with r = 0.999 for both SDZ RAD and Cs (Fig. 4 ). Calibration functions in arbitrary units were calculated: y = 277(±2)x 21(±21) for SDZ RAD; y = 578(±2)x 2723(±732) for Cs.

View larger version (23K): [in this window] [in a new window]   Figure 4. Calibration curves for (a) SDZ RAD and (b) Cs from supplemented blood. Each point represents the mean ± SD of the three samples measured in duplicate.

The intraassay CVs for 1-mL blood samples with 1, 10, and 30 µg/L added SDZ RAD were 5.7, 6.2, and 5.6%, respectively; for samples with 10, 100, and 300 µg/L added Cs, the intraassay CVs were 5.1%, 6.2%, and 5.2% (Table 3 ). Interassay CVs were 6.9%, 6.3%, and 4.9% for samples with 1, 10, and 30 µg/L added SDZ RAD, respectively, and 6.3%, 6.3%, and 5.1% for samples with 10, 100, and 300 µg/L added Cs (Table 4 ). The lower limit of detection at a signal-to-noise ratio of 3:1 was 0.05 µg/L for both SDZ RAD and Cs. The method is not compromised if the concentrations of Cs are more disparate from those of SDZ RAD. Samples containing 1 µg/L SDZ RAD plus 300 µg/L Cs could be measured accurately without any difficulty (Fig. 5 ).

View larger version (16K): [in this window] [in a new window]   Figure 5. HPLC-ESI-MS chromatogram of an extract of blood supplemented with SDZ RAD (1 µg/L) and Cs (300 µg/L).

The consecutive analysis of >500 blood samples containing SDZ RAD and Cs was achieved with a single extraction guard column and analytical column with no loss in quality. After the 4-min period for loading the extraction column, one chromatographic run took 9 min. With an interval of 1–2 min for instrument initialization, a sample turnover of four samples per hour was obtained.

	Discussion

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Using an HPLC-ESI-MS system combined with an on-line sample preparation step, we developed a reliable method for unattended routine monitoring of SDZ RAD and Cs in blood. The system combines rapid separation of the analytes by HPLC with the excellent detection performance of soft ionization by ESI-MS.

Previous work on the determination of immunosuppressive drugs by HPLC-ESI-MS (14)(15) clearly demonstrated the potential of a technique for the determination of macrolides in extracts prepared off-line by solid-phase or liquid-liquid extraction techniques. ESI-MS was the detection method of choice on the basis of the soft ionization technique suitable for unstable and highly polar compounds such as Cs and SDZ RAD. Coupling the spectrometric system to the LC system was easy, and unattended overnight operation did not raise any problems. Sensitive and specific detection of the components was achieved in the selected ion monitoring mode. Taylor et al. (15) applied tandem mass spectrometric detection to the analysis of tacrolimus in blood. Because interferences or matrix effects were not observed in our studies, use of tandem MS was not necessary for sufficient selectivity. ESI-MS detection was capable of measuring the drugs in question over the entire range of therapeutic relevance. Reproducibility and sensitivity were excellent, meeting the demands of therapeutic drug monitoring.

For the first time, we introduced on-line sample preparation preceded by manual deproteinization to a method for direct drug analysis in immunosuppressive therapy. Conventional sample preparation is the most critical step and is the most serious source of errors (22). In addition, manual off-line extraction is time-consuming and tedious. These considerations make on-line sample preparation superior to off-line procedures. The good reproducibility and accuracy of the fully automated analytical system during multiple injections made the use of an internal standard unnecessary.

The liquid chromatograph modified in our laboratory for automated sample clean-up and enrichment guaranteed continuous and rugged operation. No problems arose as the result of large volume injections of deproteinized blood by a 500-µL syringe, for which the capillary between injection valve and syringe had to be exchanged against one with wider inner diameter. All other parts of the HP 1090 autoinjector proved to be fully compatible with our demands.

Because deproteinized blood is currently the accepted matrix for the determination of immunosuppressants (9)(23)(24), the use of expensive tailor-made size-exclusion precolumn materials (25)(26) was not necessary. As with other on-line sample preparations (19)(27)(28)(29)(30), conventional C₁₈- modified silica had sufficient capacity for our purpose. In the working range studied, no overload of the precolumn was observed. Recovery was highly satisfactory. Even relatively polar compounds such as Cs and SDZ RAD were retained during the enrichment and clean-up step. As shown by blank chromatographic runs after each series of calibration runs, no memory effect related to the enrichment on the precolumn was observed. The long lifetime of the extraction guard column (>500 deproteinized 400-µL blood samples) indicates that the washing step with water was sufficient to wash out salts. Reconditioning with methanol and water was equally effective. In view of the durability of the extraction columns, the cost of consumed materials for the extraction step was negligible. Furthermore, through dovetailing the separation step and reconditioning of the guard column, re-equilibration was achieved during the chromatographic phase. In this way, chemicals and time were saved.

The analytes were eluted from the extraction column in the backflush mode, which led to narrow peaks without tailing. The chromatograms obtained were of high quality. Clean baseline resolution of the analytes was achieved within a 9-min chromatographic separation, so that automatic evaluation and integration were simple.

The results obtained show that the simultaneous determination of SDZ RAD and Cs in blood is feasible even if the working ranges differ by one order of magnitude or more. The method described outperforms traditional analytical approaches in sample turnover, reproducibility, and ease-of-use. Although photo diode array and ultraviolet detection might be applicable for high concentrations of SDZ RAD and Cs, they appear unsuitable for measurement of low trough concentrations of the compounds, especially if concentrations of these metabolites must be measured concomitantly. Laboratories tend to eliminate methods that are difficult to handle. However, frequently methods are introduced that are easy to handle but suffer from a lack of specificity (e.g., RIAs) and reproducibility. The method discussed here combines easy handling and the highest achievable accuracy, specificity, and sensitivity, if adequate instrumentation and experienced personnel are available. Thus, it is suitable universally. The system is especially well-suited for large sample series, which can be analyzed in continuous overnight operation to optimize utilization of laboratory equipment. The process is ready for measuring samples from patients routinely. It can easily be modified for a variety of other applications.

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft, grant SFB 265 A7, and the Stiftung Stipendien-Fonds des Verbandes der Chemischen Industrie e.V.

	Footnotes

 
¹ Nonstandard abbreviations: Cs, Cyclosporine; SDZ RAD 40-O-(2-hydroxyethyl)rapamycin; LC, liquid chromatography; MS, mass spectrometry; and ESI, electrospray ionization.

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