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Analytical Methodologies for the Determination of Sirolimus: A Review.

Shah Kruti V*, Chauhan SP, and Suhagia BN

Faculty of Pharmacy, Dharmsinh Desai University, Nadiad 387001, Gujarat, India

*Corresponding Author:
Shah Kruti V
Faculty of Pharmacy, Dharmsinh Desai University, Nadiad 387001, Gujarat, India.
Mobile: +91 9427614966

Received date: 11/01/2014; Revised date: 05/02/2014; Accepted date: 07/02/2014

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Abstract

Sirolimus is a macrolide lactone obtained from fermentations of a solid mold, Streptomyces hygroscopicus. Sirolimus is a potent immunosuppressive agent used to prevent rejection in organ transplantation especially useful in kidney transplants. Sirolimus was approved in 1999, by the US Food and Drugs Administration (FDA) for renal transplants and also known as Rapamycin® (RAPA). A wide variety of analytical methods have been reported for the determination of SRL in biological fluids. Methods for the measurement of Sirolimus in biological fluid have included various chromatographic methods like RP-HPLC-UV, LC-ESI-MS/MS and RP-HPLC-PDA and also included various enzymatic methods like RRA and MEIA by IMx analyzer. The applications of these methods for the determination of Sirolimus in biological sample (human or dog blood) have also been discussed in this article.

Keywords

Sirolimus (SRL), cyclosporine (CsA)

Introduction

Sirolimus (SRL) is a 31-membered triene macrolide lactone with a hemiketal masked a, b- dicarboxamide and produced by fermentation of Streptomyces hygroscopicus. It is a novel anti-rejection drug with potent immunosuppressive activity both in vitro and in vivo and also has antiproliferative activity; it is especially useful in kidney transplants.[1] It prevents activation of T cells and B cells by inhibiting their response to interleukin-2 (IL-2) and other T-cell growth factor receptors like Cyclosporine A (CsA) and Tacrolimus (TAC). SRL requires formation of a complex with an immunophilin (cystolic proteins bind with Immunosuppressive drugs), i.e. FK Binding Protein-12 (FKBP-12). However, the SRL-FKBP-12 complex does not affect calcineurin activity and inhibits a protein kinase, designated mammalian target of rapamycin (mTOR), which is a key enzyme in cell cycle progression. Inhibition of mTOR blocks cell-cycle progression at the G1 → S phase transition [2,3].

SRL is effective alone or administered in combination with other immunosuppressive agents, such as CsA. The biologic activity of SRL potentiates the immunosuppressive effects of CsA [4]. SRL was approved in mid-September, 1999, by the US Food and Drugs Administration (FDA) for renal transplants for use in combination with CsA and steroids and also known as Rapamycin® (RAPA). The available marketed formulations of LEF are enlisted in Table no. 1 [5].

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Table 1: Marketed formulations of LEF

Development and validation of analytical methods are of basic importance to optimize the analysis of drugs in the pharmaceutical industry and to guarantee quality of the commercialized product [6]. A wide variety of analytical methods have been reported for the determination of SRL in biological fluids. It includes Reverse Phase - High Performance Liquid Chromatography - UV Spectroscopy (RP-HPLC-UV), Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS), Liquid Chromatography - Electron Spray Ionization - Tandem Mass Spectrometry (LC-ESI-MS/MS) Reverse Phase - High Performance Liquid Chromatography - Photo Diode Array Detector (RP-HPLC-PDA), Radio Receptor Assay (RRA) and Micro particle Enzyme Immuno Assay (MEIA) by Tandem Mass Analyzer (MS/MS) and IMx analyzer. Among HPLC methods different internal standards, reversed phase columns with different particle size, different internal diameters and different mobile phase compositions have been used for the quantification purpose. The aim of this review is to summarize these validated techniques for the determination of SRL in biological matrix.

Various Analytical Methods Developed For Sirolimus

Biological Samples

For biological material human blood or dog blood was used. Sample-preparation is usually performed by solid-phase extraction (SPE) or liquid–liquid extraction (LLE). Methods for the measurement of SRL in biological fluid have included various chromatographic methods like RP-HPLC-UV, LC-MS/MS, LC-ESI-MS/MS andRP-HPLC-PDA and also included various enzymatic methods like RRA and MEIA by MS/MS analyzer and IMx analyzer.

Chromatographic Methods

Taylor et al. 1998 reported a HPLC- ESI- MS/MS detection method for the analysis of SRL in blood. Samples were prepared by pre-treatment with acetonitrile: 15 mM zinc sulphate (70:30, v/v) using 32-o-desmethoxysirolimus as an internal standard (IS) and C18 solid-phase extraction. The chromatographic separation was carried out on a Novapak C18 column (150 × 32.1 mm I.D., 4 μm particle size), at 50°C temperature using mobile phase consisting of methanol: 50 mM ammonium acetate buffer (pH 5.1) (80:20% v/v) at a flow rate of 0.2 ml/min. Detection was done on triple quadruple instrument using selected reaction monitoring of the mass transitions 931.8→864.6 m/z and 901.8→834.4 m/z for SRL and IS, respectively. The method validation parameters are tabulated in Table no. 2 [7]. 

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Table 2: Validation Parameters reported by Paul J. Taylor et al. 1998

Kirchner et al. 1999 developed and validated a semi-automated HPLC-ESI-MS assay for the simultaneous quantification of SRL and CsA in blood. The semi-automated sample preparation consisted of a manual deproteinization step with a mixture of methanol and zinc sulphate and automatic column switching on-line HPLC extraction on guard column (30 × 4 mm, 10 μm) filled with C18Nucleosil® 100 by using water (pH 7.0) as a mobile phase at a flow rate 0.35 ml/min. For chromatography, analytical Hypersil® ODS column (250 × 2 mm, 5 μm) as a stationary phase at 35°C temperature andmethanol: water (90:10, v/v) as a mobile phase at a flow rate 0.2 ml/min wereused. Detection was done by mass analyzer by measuring sodium adduct ions [M+Na]+ of SRL (m/z 936.6) and CsA (m/z 1224.9) with a dwell time of 0.5 s. The method result parameters are tabulated in Table no. 3. SRL and CsA were stable in blood for at least 4 months at -200C with 98% mean [8].

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Table 3: Validation parameters described by Kirchner et al. 1999

Halt DW et al. 2000 described a RP- HPLC-UV method for estimation of SRL levels in human whole blood sample by using desmethoxyrapamycin as an IS. The chromatographic separation was carried out on ultra-sphere C18 bonded silica column (25 cm × 4.6 mm, 5 μm), heated at 50°C with mobile phase consisting of acetonitrile: deionized water (65:35, v/v) at a flow rate of 1.5 ml/min by using UV detector at 278 nm. The result parameters are enlisted in Table no. 4. Samples were stable for 3 freeze-thaw cycles when stored at -20°C and for ≥2 days when stored at ambient temperature [9].

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Table 4: Validation parameters described by Halt DW et al. 2000

Maleki et al. 2000 established a RP-HPLC-UV method using desmethoxysirolimus as an IS for therapeutic drug monitoring of SRL in human whole blood. In this method, samples were prepared by extraction with l-chlorobutane and the chromatographic separation was performed on a C18 column (4.6 mm x 15 cm, 3 pm) using mobile phase consisting of methanol: acetonitrile: water (68:2:30, v/v/v) with flow rate of 1.0 ml/min at temperature 60°C and detected by UV detector at 278 nm. The validation parameters are tabulated in Table no. 5 [10].

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Table 5: Validation parameters reported by Maleki S et al. 2000

Cattaneo et al. 2002 presented RP-HPLC-UV method for the analysis of SRL in whole blood using 32-o-desmethoxyrapamycin as an IS. Here, the samples were purified by zinc sulfate and then extracted with acetone followed by solid-phase extraction. The chromatographic separation was performed on a column packed with Ultra sphere C8 (7534.6 mm, 3 mm) and heated at 50°C by using a mixture of distilled water: methanol: acetonitrile (34:30:36, v/v/v) at a flow rate of 1 ml/min and detected by UV detector at 278 nm. The method result parameters are tabulated in Table no. 6 [11].

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Table 6: Validation parameters reported by Cattaneo et al. 2002

Campanero MA et al. 2004 established RP-HPLC-PDA method for TDM of SRL in blood samples from renal, cardiac and hepatic transplants. Here, SRL and Desmethoxyrapamycin (IS) samples were purified by a combination of a precipitating blood matrix with zinc sulphate and a single step liquid-liquid extraction with acetone and 1-chlorobutane. The Separation was carried out at 50°C on a C18 column (150 × 2.1mm, 5μm), in isocratic mode using mobile phase consisting of distilled water: methanol: acetonitrile (26:50:24, v/v/v), at a flow rate of 0.25 ml/min and detected by UV diode array detector at 278 nm. The method validation parameters are tabulated in Table no. 7 [12].

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Table 7: Validation Parameters reported by Campanero MA et al. 2004

Lee et al. 2010 proposed LC-MS/MS method for determination of SRL in dog blood by using Tacrolimus (TAC) as an IS. Here, the concentration of SRL was quantified in blood samples for up to 36 h after the dog had received a 3 mg/kg dose of SRL. The chromatography was achieved on a Prodigy Phenyl-3 column (2.0mm × 50mm, 5μm), in isocratic mode using mobile phase composed of acetonitrile: methanol: ammonium bicarbonate (10 mM) (68:17:15, v/v/v) at a flow rate of 0.25 ml/min and detection was carried out by triple quadrupole mass spectrometer for optimizing multiple reaction monitoring (MRM) conditions of the analytes in positive ion mode. Here, precursor→product ion transition for SRL (m/z931.7→864.5) and IS (m/z 821.6→768.5) were monitored. The method result parameters are tabulated in Table no. 8 [13].

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Table 8: Validation parameters reported by Lee et al. 2010

Mano et al. 2011 established LC/ESI–MS/MS method for quantitation of SRL in human whole blood. For sample preparation, first pre-treatment was done with a zinc sulphate protein precipitation, an extraction using octadecylsilyl-silica gel for eliminating water-soluble and hydrophilic compounds, and Hybrid SPE Cartridge treatment to eliminate phospholipids. The separation was performed using Ascomycin as an IS on a Capcell Pak MG II (150mm×2.0mm i.d., 5μm) at 50°C column temperature using 10mM ammonium acetate: methanol (20:80, v/v) as a mobile phase at a flow rate of 0.2 mL/min. Detection was done by selected reaction monitoring (SRM) at the transitions of m/z 931.6–864.5 and m/z 809.5–756.5 were used for monitoring SRL and Ascomycin, respectively, and dwell time was 500 ms. The method validated parameters are tabulated in Table no. 9. Sample was stable for at least 46 days under -200C storage for at least three freeze-thaw cycles [14,15] .

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Table 9: Validation Parameters described by Mano et al. 2011

Anil Kumar et al. 2012 performed analytical method RP-HPLC with PDA detection for quantification of SRLin blood samples using ketoconazole (KTZ) as IS. Extraction was performed using dichloromethane under nitrogen atmosphere andseparation was accomplished by waters Xterra MS C18 analytical column (250 × 4.6 mm, 5μm), in isocratic mode using mobile phase consisting of methanol: water: glacial acetic acid (90:10:0.1%, v/v) at a flow rate of 1 ml/min and detected by PDA detector at 278 nm. The method validation parameters are tabulated in Table no. 10 [16].

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Table 10: Validation Parameters reported by Anil Kumar et al. 2012

Enzymatic Methods

Goodyear N. et al. 1996 described a RRA for estimation of SRL in whole blood. for this, SRL was resuspended from the dried tubes with [3H]-dihydro FK506 at 160,000 disintegrations per min (dpm) followed by direct methanol extraction, using 15-25 μg of protein containing the 14 or 52 kDa protein for final diluent. After this, samples applied to Sephadex LH-20 columns, bed volume 1.8 mL previously equilibrated with LH-20 buffer (20 mmol/L Tris (pH 7.2), 5 mmol/L β-mercaptoethanol, and 0.5 g/L sodium azide), and eluted with 150 μL followed by 1.25 mL LH-20 buffer to separate free [3H]-dihydro FK506 from bound and the bound portion was measured using a scintillation counter with a counting efficiency of 59%. Nonspecific binding was estimated using a 1 mg/L concentration of unlabeled SRL. The validation parameters are tabulated in Table no. 11. In interference study, no interference observed in RRA for SRL with CsA, Methotrexate, Dexamethasone and Prednisolone [17].

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Table 11: Validation parameters by Goodyear N. et al., 1996

Jones K et al. 2000 studied a MEIA for prepared sample measurement in whole blood and HPLC-MS/MS method used for SRL estimation. In this method, samples were prepared by protein precipitation extraction technique. Specificity was determined by the addition of 2 sirolimus metabolites (Hydroxysirolimus and 4l-o-demethylsirolimus) to sirolimus free human whole blood. Method validation parameters are tabulated in Table no. 12. In stability study, no loss of SRL during a total of 3 freeze-thaw cycles and samples were stabled for 10 days at 40C and at ambient temperature (22°C protected from light) [18].

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Table 12: Validation Parameters by Jones K et al. 2000

Wilson D et al. 2006 described microparticle enzyme immunoassay (MEIA) for sirolimus in whole blood by using IMx analyser. In this method, MEIA was a one-step competitive assay utilizing a murine monoclonal antibody covalently coupled to carboxylated micro particles. Here, samples were pretreated with a methanolic precipitating reagent and the IMx probe/electrode assembly combines pre-treated sample, micro particles, and conjugate to the incubation well of the disposable MEIA reaction cell. After this, it is reacted with alkaline phosphate and formed substrate, 4-Methylumbelliferyl phosphate, is converted to a fluorescent product which is measured by the IMx optical assembly. For Specificity, SRL was metabolized into primarily hydroxylated and/or demethylated forms by the CYP3A4 enzyme system. Method result parameters are tabulated in Table no. 13 [19].

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Table 13: Validation Parameters by Wilson D et al. 2006

Conclusion

This review presents the specific, sensitive and accurate analytical techniques for the estimation of SRL in blood are based on the separation by HPLC with different detectors and some enzymatic methods. Spectrophotometric, spectrofluorimetric and Chromatographic methods other than HPLC are not reported for SRL quantification in both pharmaceutical preparations and biological fluids. However in future some newer aspects can be thought for the development and validation of stability indicating method as well as degradation kinetic study and impurity study.

References

http://www.encyclopaediaislamica.com/