Quantification of Immunosuppressant’s in Blood using LC-MS/MS

Review Article

Austin Chromatogr. 2016; 3(1): 1039.

Quantification of Immunosuppressant’s in Blood using LC-MS/MS

Al-Jenoobi FI¹, Alam MA²* and Al-Mohizea AM³

Department of Pharmaceutics, King Saud University, Saudi Arabia

*Corresponding author:Alam MA, Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O.Box 2457, Riyadh 11451, Saudi Arabia

Received: December 07, 2015; Accepted: February 25, 2016; Published: March 01, 2016


Numerous analytical advancements have been introduced for Therapeutic Drug Monitoring (TDM) of immunosuppressant’s (cyclosporine a, tacrolimus, everolimus and serolimus). In recent years, liquid chromatography in tandem mass spectrometer has been the technique of choice for the determination of immunosuppressants. The automated on-line sample processing and purification technologies have simplified the sample purification and extraction process. In present review, the important liquid chromatographic as well as mass spectrometric parameters which can influence the determination of immunosuppressants are also discussed. It has been observed that extraction procedure, mobile phase composition, mobile phase gradient, column type, column temperature, cone voltage and analyte adduct ions have direct impact on the determination of immunosuppressants. Dried blood spot sampling for TDM of immunosuppressants has been suggested by several researchers. Present review also briefed some commercial analytical kits for immunosuppressants. On the basis of available literature, it was concluded that LC-MS/MS is the gold standard analytical technique for therapeutic monitoring of immunosuppressants.

Keywords: Immunosuppressants; Bio-analysis; LC-MS/MS; Cyclosporine; Tacrolimus; Everolimus; Sirolimus


LC: Liquid Chromatography; MS/MS: Mass Spectrometer; LCMS/ MS: Liquid Chromatography in Tandem Mass Spectrometer; ESI: Electrospray Ionization; UPLC: Ultra-Performance Liquid Chromatography; TDM: Therapeutic Drug Monitoring


The immunosuppressants such as cyclosporine A (Neoral®), tacrolimus (Prograf®), sirolimus (Rapamune®) and everolimus (Zortress®) have been indicated for the prophylaxis of organ rejection in patients receiving allogeneic organ transplants. The cyclosporine, tacrolimus, sirolimus and everolimus are poorly soluble in water. Their oral absorption is incomplete and has poor bioavailability. They are extensively metabolized by CYP3A isoenzyme system. Cyclosporine A primarily eliminated through biliary route. Fecal elimination predominates for tacrolimus, sirolimus and everolimus. All these four immunosuppressants extensively bound plasma proteins. The immunosuppressive action of these substances is primarily due to parent drug.

These aforesaid immunosuppressants are categorized as narrow therapeutic indexed drug substances. Their blood concentration below therapeutic level may leads to organ rejection (therapy failure) and the concentration above therapeutic level will be toxic. Therapeutic drug monitoring is recommended for all patients receiving treatment with any of these immunosuppressants. Over the years numerous analytical methods have been developed for the analysis of these immunosuppressants in biological fluids for pharmacokinetic studies and therapeutic monitoring. Many of are methods which can analyze all four drugs simultaneously. The analytical methods for determination of these immunosuppressants should be accurate, precise, sensitive, highly selective and robust to avoid any wrong determination. The analysis of cyclosporine, tacrolimus, sirolimus and everolimus is a complex process, since these drugs are analyzed in biological fluids such as blood, plasma, urine and aqueous humour in very low concentrations; usually in ng/ml. Different types of detectors (e.g. ultraviolet detectors, mass spectrometers and electrochemical detectors etc.) have been used in liquid chromatographic methods. Liquid chromatography in tandem mass spectrometer has been considered as gold standard technique for the analysis of immunosuppressants in biological tissues and fluids. Nowadays antibody-based immunoassays for therapeutic drug monitoring are being replaced by liquid chromatography tandem mass spectrometer. The immunoassays have been reported to overestimate the cyclosporine A concentration in blood because of the cross reactivity of cyclosporine A metabolites [1,2]. Schutz et al. reported that in a liver recipient group CEDIA and the AxSYM showed a mean overestimation of CsA by 43% and 47%, respectively [1]. The other disadvantages of immunoassay methods are their high cost as compare to liquid chromatographic methods and their inability to simultaneously measure the two or more immunosuppressant’s [3]. The liquid chromatographic techniques, especially LC-MS/ MS provide higher analyte specificity, when compared with immunoassays. Simultaneous analysis of these immunosuppressants has been carried out by LC-MS/MS methods.

Hermann et al. enlisted the chromatographic parameters used in previously published papers for the estimation of cyclosporine A and its metabolites. The different investigators have used different set of chromatographic conditions: the column temperature varies between 50-80°C, the most commonly used columns are C8 and C18, the flow rate varies between 0.3-2.5 ml/min, the range of wavelength of detection was 206-230nm and the most common organic modifiers were Acetonitrile and methanol [4].

Biological samples

In pharmacokinetic investigations the immunosuppressants have been measured in various biological fluids. However, blood is the preferred biological fluid for therapeutic monitoring of immunosuppressants. There are several factors which make the blood as the preferred sampling fluid. One of these factors is: the high affinity of immunosuppressants for cellular components of blood. The higher concentrations of immunosuppressants have been observed in blood compared to plasma, so the blood is recommended as the commonly accepted biological matrix for their estimation [5,6]. Cyclosporine A is highly bound to red blood cells and plasma proteins; and during storage its distribution between red blood cells and plasma or serum is temperature dependent [7,8]. Therefore, the determination of cyclosporine A is preferred in whole blood to minimize the temperature dependent variations of cyclosporine A concentrations [8]. Koster et al. investigated that varying hematocrit value and analyte concentration influenced the extraction recoveries of sirolimus and everolimus; while there was no significant effect on the extraction recoveries of cyclosporine and tacrolimus [9]. Koster et al. also observed the positive as well as negative bias in the results when compared with the standardized hematocrit value (0.35 L/L). At the lowest hematocrit value (0.20L/L) and highest drug concentration (50μg/L) the sirolimus and everolimus showed -20% and -28% bias, respectively [9]. There are several analytical methods which can measure the immunosuppressants in other biological fluids. The cyclosporine A can be determined in blood, plasma, serum, bile and urine [10,11]. Szerkus et al. determined cyclosporine A in tissues (cornea, conjunctiva and eye globe) and fluids (lacrimal fluid and aqueous humor) of rabbit eye [12]. Guada et al. developed and validated an ultra-high performance LC-MS/MS method for the quantification of cyclosporine A in lipidnano-systems and mouse biological matrices such as whole blood, kidneys, lungs, spleen, liver, heart, brain, stomach and intestine; and the method was successfully applied for pharmacokinetic and biodistribution studies of cyclosporine A [13]. Belostotsky et al. investigated the tacrolimus distribution in the blood and saliva; and found a poor correlation between the drug levels in both biological fluids. In some of the cases the tacrolimus level was not detectable in saliva though the drug was present in the blood sample. Because of this poor correlation the sampling of saliva can’t be considered for therapeutics monitoring of tacrolimus [14].

Amini and Ahmadiani reported that the interference increases in frozen blood samples and the number and intensity of interfering peaks gradually increased after long storage at 4 °C [15]. Several stability studies suggested that cyclosporine A, tacrolimus, sirolimus and everolimus can be measured in blood samples without stability concerns, if handled and stored properly. All these four immunosuppressants have been found stable in blood sample at room temperature for few days and at -80 °C for several months. The extracted samples (acetonitrile and methanol) were also stable in autosampler for a sufficient period of time (24 hrs to few days). The cycloaporine A is stable in blood samples for 2-3 months when stored at 1-4 °C [15].

Dried blood spot

Generally, the therapeutic drug monitoring of immunosuppressants is performed in venous blood samples. Recently, several investigations have been carried out on dried blood spot sampling method and it has been suggested as an alternative sampling method. Dried blood spot sampling is a newly evolving idea for therapeutic monitoring of immunosuppressants [16-21]. The dried blood spot sampling has several advantages over the conventional (venous) blood sampling. The patients can withdraw the capillary blood themselves using an automatic lancet. The first drop of blood is discarded and the second drop is applied to the sampling paper. To ensure the accuracy and uniformity of the method, the circles on the sampling paper are homogeneously and symmetrically filled with blood drop. The blood drop is dried on the sampling paper at room temperature and sealed packed properly. For extraction, the paper disks with a predetermined diameter (~ 8 mm) are punched out with the help of a whole puncher [16]. The dried blood spot method offers several advantages including small samples size (10-50 μL, preferably 30 μL bloods is applied as spot) and fewer hospital visits, since the sample can be posted to the laboratory [22-24]. Hinchliffe et al. developed a UPLC-MS/MS method for simultaneous estimation of cyclosporine A and tacrolimus from dried blood spot [23]. Rao et al. developed and validated a high-throughput LC- ESI-MS method for monitoring the sirolimus on a dried blood spot [24].

Wilhelm et al. and Hinchliffe et al. perform validation studies in which they compare the dried blood spot sampling methods with venous sampling method. In dried blood spot sampling method capillary blood was obtained from a finger prick with an automatic lancet and the drop of blood ( ~30 μL) was applied to sampling paper; while venous blood sampling was performed by venipuncture. The conclusions suggested that dried blood spot sampling method can be used as an alternative for conventional venous blood sampling in therapeutic monitoring of immunosuppressants [25,26]. Wilhelm et al. studied the influence of the hematocrit value on cyclosporine A concentration in dried blood sample, and observed that the difference in hematocrit value influences the analysis of cyclosporine A in the dried blood sample. The blood samples of high hematocrit value have high viscosity, and because of high viscosity the blood distribution on the paper is poor. The blood sample with higher hematocrit value (higher viscosity) will form smaller spot and center of the spot will be thick and have more drug concentration [27]. The perfect round spots with uniformity can be produced by spotting the blood in a fixed volume on the paper. The spots made with small volume of blood may influence the results if it is not sufficient to reach the margins of the fixed volume circle and the punched out part will show the white edges of unspotted paper [21]. Dried blood spot samples of everolimus were found stable for 34 days at 32 °C and for 3 days at 60 °C, while at 70 °C the everolimus was suggested unstable [19]. Sadilkova et al. investigated the stability of patient dried blood spot samples. Tacrolimus and cyclosporine A were found stable for at least five days at the temperature up to 60 °C, while degradation was observed in sirolimus at 60 °C within 24 h [20].

Extraction procedures

The protein precipitation and liquid-liquid extraction techniques have been used for extracting the immunosuppressants from biological fluids. Protein precipitation is the most commonly used technique, since it is simple and involve less cost. In protein precipitation technique blood sample has been mixed with ZnSO4 solution and mixed properly by vortexing the mixture. The organic solvents such as methanol (MeOH) and Acetonitrile (ACN) are widely accepted protein precipitants. The ZnSO4 solution has been added to whole blood sample to rupture the red blood cells and as a result to release the bound drug. Koster et al. found that use of ZnSO4 provides good process efficiency for cyclosporine and tacrolimus while a poor efficiency was observed for sirolimus and everolimus [28]. Some of the protein precipitation based extraction methods for sirolimus and everolimus have used ZnSO4 solution in extraction procedure [3,29]. In general, the protein precipitated samples are centrifuged and the supernatant is removed and directly injected for the analysis. These protein precipitated samples can further be cleaned by loading and eluting on a solid phase extraction cartridges. In Solid Phase Extraction (SPE) the extraction cartridge (or extraction column) is conditioned with Solvent (s) and or buffer and then loaded with the supernatant of protein precipitated sample. Extraction column is flushed with the solvent system followed by analytes extraction with a suitable solvent system, and then extracted sample is injected into analytical column. The solid phase extraction can also be performed on-line. For on-line purification the liquid chromatography system is supported with multiport switching valve. The supernatant sample is injected into extraction column with suitable solvent system and then flushed at high flow rate. After flushing the switching valve is changed and the analytes are eluted from extraction column to the analytical column by using solvent system of higher organic phase. The supernatant of protein precipitated samples also has been cleaned by using turbulent flow chromatography (Cyclone 50 x 1 mm polymeric column, Cohesive Technologies, USA) supported with switching valve [30]. Numerous other advancements have been introduced in the sample extraction procedures. The automated and semi-automated online extraction procedures were developed for the extraction of immunosuppressants from blood. McMahon et al. uses semi-automated solid-phase extraction while Brignol et al. uses semi-automated high-throughput liquid/liquid extraction procedure for the extraction of cyclosporine A and everolimus [31,32]. Alvarez and Weiner purify the extracted supernatant of protein precipitated plasma samples on-line and directly injected into the LC-MS system [33]. Marinova et al. developed an automated protein precipitationbased whole blood sample preparation protocol for the quantification of sirolimus, everolimus and tacrolimus by using a liquid handling system [Tecan Freedom EVO 150 liquid handling platform (Tecan, Männedorf, Switzerland) equipped with an 8-channel liquid handling arm with standard fixed tips, one robotic plate-handling arm and one plate shaker (Te-Shake), and an additional component vacuum separation module (Te-VacS)] [29]. The supernatants of protein precipitated samples were then subjected to on-line solid phase extraction using column switching technique [29]. Hassell et al. demonstrated the application of Prelude TM Sample Preparation Liquid Chromatography (SPLC) system connected with a TSQ Vantage TM triple stage quadrupole mass spectrometer for measuring immunosuppressant’s (cyclosporine A, tacrolimus, serolimus and everolimus) and chemotherapeutic drugs. The samples were cleaned online by turbo flow technology and the analytical separation was carried out on Prelude TM SPLC system [34]. MagSi- TDMPREP (amsbio) is a fully automated sample preparation kit. The kit eliminates the interfering proteins, phospholipids and salts from whole blood sample. The process of MagSi-TDMPREP involves the lysis of whole blood cells followed by addition of internal standard. The MagSi-TDMPREP beads are added to the sample and then protein precipitant is added. The precipitated protein are bound to the beads and removed by using magnetic separation. The supernatant can be directly injected into the LC-MS/MS [35,36].

Internal standards

For the analysis of drugs extracted from biological samples, it is preferred that internal standard and analyte should have physicochemical similarities [37]. Because of physicochemical similarities the internal standard and analyte will be extracted uniformlyin a simple solvent system and by a common procedure. The extraction efficiency of compounds having similar physicochemical properties will also be closely related. Further it may also simplify the elution of both compounds on a same type of analytical column with less complex mobile phase composition. The similarities or closeness in chemical properties (such as molecular weight and chemical structure) of analyte and internal standard also allows working on almost similar mass spectrometric parameters such as cone voltage, collision energy, type of ion adducts etc. For immunosuppressants estimation in biological fluid by LC-MS/MS, the most preferred internal standards are their isotope-labeled compounds (isotopelabeled compound viz. evelolimus-d4, sirolimus-d3, cyclosporine A-d4). The cyclosporine D also has been preferred as internal standard for the estimation of cyclosporine A. The other used internal standards include ascomycine, amiodarone, desmethoxyrapamycin and desmethyl sirolimus.


For routine therapeutic monitoring of drug substances a sensitive, selective and rapid assay is required. The liquid chromatography (HPLC or UPLC) in tandem Mass Spectrometer (MS/MS) has been the technique of choice for pharmacokinetic investigations and therapeutic monitoring of immunosuppressant’s.

Liquid chromatography: Over the years, several advancements have been introduced in the liquid chromatographic systems. The Ultra-Performance Liquid Chromatography (UPLC) is one of the recent technological advancements in the analytical field. UPLC analyzes the drugs in a shorter time when compared with traditional HPLC. Pedraglio et al. compared the performances of HPLC-MS/ MS and UPLC-MS/MS in terms of sensitivity and resolution. UPLC significantly reduces the analysis time, decreases the mobile phase consumption, increases sensitivity and resolution [38,39]. Hsieh et al. developed an Ultra-Performance Hydrophilic Interaction Liquid Chromatography (UPHILIC) tandem mass spectrometer for the determination of everolimus in mouse plasma [40].

The C18 is the most commonly used analytical column for the elution of immunosuppressants. Amini and Ahmadiani suggested that C18 and C8 columns should be operated with higher percentage of acetonitrile (75%) at a temperature of 70 °C, while CN or TMS columns can be operated at lower temperature (60 °C) and lower acetonitrile ratio (50%) [15]. Baldelli et al. reported a chromatographic method where analytes were eluted on C8 column (at 60 °C) for over 36 min (everolimus 9 min, cyclosporine A 21.3 min and cyclosporine D 32.2 min) by using a mobile phase consisted of 56% acetonitrile in water [41]. Apart from particle packed columns the cyclosporine A and sirolimus have been successfully eluted on monolithic column [33,24]. Rao et al. observed that monolithic column (Merck Chromolith Performance RP18e (25 x 4.6 mm)) generates much less back pressure (25 kg f) than the particulate column (444 kg f), while maintaining its efficiency [24]. Hatsis and Volmer demonstrated the applicability of cyano columns for simultaneous estimation of tacrolimus, sirolimus and cyclosporin A. The drugs were analyzed using selected reaction monitoring in negative electrospray ionization mode on a triple quadrupole mass spectrometer. According to Hatsis and Volmer, at 50 °C the chromatographic efficiency (theoretical plates per column) of tacrolimus, sirolimus and cyclosporin an on cyano column was much better than that on C18 column [42]. The sensitivity/ limit of detection in negative ionization mode has been reported to be higher than the positive ionization mode, with an argument that there are fewer product ions in negative ion mode than in positive ion mode [42].

The resolution and peak shape of cyclosporine A is significantly influenced by changing the column temperature [43]. Sawchuk and Cartier have optimized the column oven temperature. They also suggested that column life is shortened by increasing the temperature of column oven [43]. The tips for column longevity were suggested by Lensmeyer and Fields. The column life can be improved by careful cleaning the column and continuously maintaining the flow of mobile phase, and by using silica saturated column at lower column temperature, shorter run time and by using pre-column [44,45]. Ouyang et al. developed a liquid chromatographic method which can analyze the cyclosporine a in human blood at room temperature. The methods is said to improve detection limit, column life and peak broadening was also controlled. A mobile phase composition comprising isopropanol: acetonitrile: water (80:10:10) and flowing at 1ml/min is said to improve peak shape at column temperature of 25 °C [46]. Salm et al. developed a rapid LC-MS analytical method for the determination of CsA with a very short retention time between 0.5-1.0 min and total run time of 2 min [47]. The analytical column life was suggested at least 500 injections. Since the temperature of column oven was high (70 °C) so the limited column life is promised [47].

Hermann et al. evaluated the importance of essential chromatographic parameters such as percentage of organic modifier, column oven temperature, flow rate, pH of mobile phase and gradient steepness of mobile phase; in the determination of cyclosporin A and its metabolites, AM1, AM9 and AM4N. All parameters (except HCl addition) were found to be of significance to one or more response variables (resolution between AM1 and AM9, retention time of AM1/ AM9 and retention time of cyclosporine A) [4]. For the separation of analytes, the optimal chromatographic parameters were determined as: a reversed phase C8 column maintained at 80 °C, and mobile phase containing acetonitrile and water flowing in linear gradient (ACN 52.8 to 73%) at the rate of 0.8ml/min [4].

The preferred mobile phase composition for elution of immunosuppressants includes methanol and or acetonitrile as organic modifier and ammonium acetate buffer as aqueous medium. Formic acid in low percentage also has been used in mobile phase composition. In LC-MS/MS methods of immunosuppressants the mobile phase was pumped in gradient mode, though some isocratic LC-MS/MS methods were also developed.

Adducts: In LC-MS/MS methods the mass detector quantifies the analyte ions according to their mass/ charge (m/z) ratio. There are several modes and source of ionization in different mass spectrometers. Positive electrospray ionization is the commonly used mode of ionization for the analysis of small drug molecules. Generally, the positive electrospray ionization mode produces protonated species [M+H]+. However for some molecules the formation of alkali metal ion adduct such as sodium-adduct [M+Na]+ or potassium-adduct [M+K]+ has been reported. The formation of these adducts depends upon the characteristics of the analyte molecules, sample processing, mobile phase composition and mass operating parameters. The alkali adducts are difficult to fragments into small species (daughter ions or fragments) and are unsuitable for selected reaction monitoring. Nozaki et al. reported an on-line ion suppression technique to eliminate alkali metal ion adducts. The tacrolimus solution was passed through ion suppressor device (ion chromatography system with a cation-exchange resin), and then directly introduced into the ion-trap mass spectrometer [48].

Different adducts for the same analyte have been observed under different solvent compositions. The direct infusion of methanol solution of sirolimus and tacrolimus showed prevalence of their sodium adducts, while the presence of mobile phase (methanol and ammonium formate buffer 10 mM, pH 3.0) in full scan mass spectrum showed dominance of ammoniated adducts of cyclosporine, sirolimus, and tacrolimus [49]. Chan et al. reported that isomeric active metabolites (13-O-demethylated (M1), 31-Odemethylated (M2) and 15-O-demethylated (M3) Tacrolimus) of tacrolimus demonstrated a strong ability to form ammonium-adduct and sodium-adduct ions. The sodium-adduct ions of the metabolites were easily formed in ion source but they were not found suitable for multiple reaction monitoring because of their poor fragmentation. So the ammoniumadducts were selected as precursor ions for multiple reaction monitoring [50]. The fragmentation of sodium adduct of everolimus is poor when compared with the fragmentation of ammoniumadducts of everolimus [51]. Sodium adduct of everolimus has been measured as single ion monitoring [52,53].The high intensity signal of sodium adducts of sirolimus and its metabolites were monitored in positive electrospray ionization mode [6]. Koseki et al. run a mass spectrum of cyclosporine A and its metabolites (AM1, AM4N, and AM9) in atmospheric pressure chemical ionization and in positive electrospray ionization mode. The sodium and ammonium adducts of cyclosporine A and its metabolites were observed in positive electrospray ionization mode, while the protonated ion peaks of cyclosporine A and its metabolites were observed with strong signal response in atmospheric pressure chemical ionization mode [54].

The several adducts of immunosuppressants have been analyzed. The ionic adducts which can be fragmented into daughter ions can be analyzed easily by using multiple reactions monitoring mode, while the hard adduct ion do not fragments easily into stable daughter ions and are directly analyzed in single ion recording mode. The commonly analyzed adduct for cyclosporine a, tacrolimus, sirolimus, and for everolimus is ammonium adduct. Volosov et al. reported that the use of ammonium acetate induced production of ammonium adducts [NH4+] of the analytes (cyclosporine A, tacrolimus, sirolimus), but the ammonium adduct of cyclosporine A (m/z 1220) could not produce a stable abundant fragment of cyclosporine. So the intact cyclosporin ion (m/z 1202.7) was monitored as a “fragment” of its own ammonium adducts [55].

Citation: Al-Jenoobi FI, Alam MA and Al-Mohizea AM. Quantification of Immunosuppressant’s in Blood using LC-MS/MS. Austin Chromatogr. 2016; 3(1): 1039. ISSN 2379-7975