Volume 2 • Issue 2 • 1000118J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal

Open AccessResearch Article

Babu et al. J Anal Bioanal Techniques 2011, 2:2 DOI: 10.4172/2155-9872.1000118

Keywords: Tacrolimus; Rat whole blood; Triple quadrupole LCMS;Pimecrolimus; Sodium citrate

IntroductionTacrolimus is an immunosuppressive agent (Figure 1) isolated from

streptomycin tsukubaensis [1]. It is a macrolide lactone with a hemik-etal masked α, β-di-ketoamide and a molecular mass of 803.5 Da [2]. It is the basis of immunosuppressive drug regimens after liver & kidney transplantation & it has also been used for heart, pancreas, bone mar-row, small bowel, lung transplant & for the treatment of T-cell medi-ated autoimmune diseases such as allergic encephalomyelitis [1,3].Therapeutic drug monitoring is recommended for monitoring trough blood/plasma tacrolimus levels as an aid in adjusting the dose to pre-vent rejection with toxicity. Two commercially available assays are used to monitor tacrolimus levels, especially in whole blood. Abbott 1Mx® & PRO-TRACTM by incstar [4-6]. In general, clearance and volume of distribution are higher when calculated using plasma drug concentra-tions than when whole blood is used. This is because drug concentra-tions are significantly higher in the whole blood as results of extensive drug binding to erythrocytes in patients with transplants [7]. Whole blood is recommended medium than plasma for assessing the phar-macokinetics of tacrolimus[8].The average bioavailability of tacrolimus was reported to be approximately 20-25% in healthy human subjects [9-10].Tacrolimus is a potent substrate of cytochrome P450 and CYP3A4 enzymes efficiently metabolize tacrolimus in the small intestine as well as in the liver [11,12]. Many studies have suggested that tacrolimus is a potent substrate of P-glycoprotein (P-gp) and that an active secretory transport mediated by intestinal P-gp is largely responsible for the low oral bioavailability of tacrolimus [13-17].

A survey of the literature indicates that immunoassays often lack specificity, especially for drugs which are extensively metabolized, the cross-reactivity affecting some immunoassays more than others [18–21]. For tacrolimus and cyclosporine the cross-reactivity of the metabo-lites causes a very significant (up to 60%) and unpredictable overesti-

mation of the concentrations [20–23]. Assay techniques that provide specific tacrolimus concentration measurement with greater sensitivity, such as liquid chromatography–tandem mass spectrometry (LC–MS/MS) are now widely employed [24–28].

In recent years, a number of laboratories have reported for the use of high throughput bioanalytical procedures using LC-MS-MS. So our method is simple, rapid, sensitive, specific, robust and novel that makes it an attractive procedure in high throughput.

ExperimentalReagents

The pure substances of tacrolimus with pimecrolimus (internal standard; IS) were obtained from Biocon India. Chemical structures are presented in (Figure1) Stock solutions of tacrolimus (1mg/mL) & IS (1mg/mL) were separately prepared in 5mL volumetric flasks with methanol. HPLC grade methanol from JT Baker, t-butyl methyl ether & ammonium acetate were from Merck (Worli Mumbai India), HPLC type 1 water from milli Q system (Millipore, Bedford.MA USA) was used and blank rat whole blood from healthy albino Wistar rats was obtained from blood service centre of Bioneeds, Bangalore (India).

*Corresponding author: A.R.Suresh Babu, Department of Bioanalytical, G7Synergon Pvt Ltd, No537, 9th cross, 5th main, Tatanagar, Sahakaranagar post, Bangalore-560092, India, Tel: +91 080-22172700; E-mail: arsureshbabu@rediffmail.com, sureshbabu.ar@g7synergon.in

Received January 31, 2011; Accepted March 26, 2011; Published March 30, 2011

Citation: Babu ARS, Thippeswamy B, Vinod AB (2011) Determination of Tacrolimus in Rat Whole Blood Utilizing Triple Quadrupole LC/MS. J Anal Bioanal Techniques 2:118. doi:10.4172/2155-9872.1000118

Copyright: © 2011 Babu ARS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

AbstractA simple, sensitive and rapid method was developed for quantitation of tacrolimus in rat whole blood utilizing

triple Quadrupole LC/MS. An aliquot of 0.1 mL of whole blood sample was extracted with t-butyl methyl ether using a Heidolph vortexer. The chromatographic separation was performed by using chromolith fast gradient HPLC RP 18e (2mmX50mm.i.d) column with a mobile phase of 90% methanol and 10% 2mM ammonium acetate buffer followed by MS/MS detection. The analyte was quantitated in negative ionization mode. Multiple reaction monitoring (MRM) using the transition m/z 802.4→560.2 and m/z 808.4→548.6 was performed to quantify tacrolimus with IS (pimecrolimus), respectively. The method had a total chromatographic run time of 2.5 min; and linear calibration curves over the concentration range of 20.931 -1000.703 ng/mL.The lower limit of quantification (LLOQ) was 20.931 ng/mL. Use of sodium citrate (3.85%) as an anticoagulant in rat whole blood and samples were stable for at least the time required to assay the number of samples that could be placed in the auto sampler which is maintaining temperature of 10°C. The between and within batch precision and accuracy of the method were determined by using 6 quality control samples. The highest %CV 478.908 ng/mL (8.01% within run & 3.07 between run), with other %CV<5%. The recovery ranged 23.92% for tacrolimus over range of 50.285 to 798.179 ng/mL and was 18.52% for pimecrolimus respectively. The validated method was successfully applied to the quantification of tacrolimus concentration in rat whole blood.

Determination of Tacrolimus in Rat Whole Blood Utilizing Triple Quadrupole LC/MSA. R. Suresh Babu*, B. Thippeswamy and A. B. Vinod

A.R.Suresh Babu, Department of Bioanalytical, G7Synergon Pvt Ltd, No537, 9th cross, 5th main, Tatanagar, Sahakaranagar post, Bangalore-560092, India

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Volume 2 • Issue 2 • 1000118J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal

Citation: Babu ARS, Thippeswamy B, Vinod AB (2011) Determination of Tacrolimus in Rat Whole Blood Utilizing Triple Quadrupole LC/MS. J Anal Bioanal Techniques 2:118. doi:10.4172/2155-9872.1000118

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Apparatus / instrumentation

Chromatographic analysis was performed using an Agilent 1200 Series LC System (Agilent technologies, INC; VBA) equipped with G1312B Binary pump, G1315C UV detector, G1316B thermostatic column compartment, G1329B auto sampler equipped with a G1330B thermostat. Mass spectrometric detection was performed in Triple Quadrupole LCMS 6410 instrument with Agilent technologies USA us-ing MRM. A turbo electrospray interface in negative ionization mode was used. Mass data acquisitions with integration were controlled by Agilent mass hunter chemstation (B.01.03) software.

LC-MS/MS conditions

Chromatography was performed on Chromolith Fast Gradient RP 18e HPLC Column (2x50mm), maintained at 40ºC. The mobile phase composition was 2mM ammonium acetate buffer: methanol 10:90 v/v (Binary Flow) which was pumped at flow-rate of 0.2 mL/min without splitter. The auto sampler temperature was set at 10ºC. The main work-ing parameters of the mass spectrometer are summarized in Table 1.

Preparation of standard and quality control (QC) samples

Stock solutions of tacrolimus at a concentration of 1mg/mLwere prepared by dissolving the accurately weighed reference substance in methanol. The stock solution was then serially diluted with metha-nol water (50:50v/v) to give working solutions at the following con-centrations: 20.931, 41.861, 49.835, 99.670, 240.169, 480.337, 800.562 and1000.703 ng/mL. The other stock solution was independently dilut-ed in a similar way to achieve QC solution at concentrations of 21.120 (LLOQ), 50.285 (low), 478.908 (medium) and 798.179 ng/mL (high). Internal standard working solution (5 µg/mL) was prepared by dilut-ing the 1mg/mL stock solution of pimecrolimus with methanol water (50:50v/v). All the solutions were kept at 4ºC -8ºC and were brought to room temperature before use.

Both the calibration standard samples and the quality control sam-ples which were used in the pre-study validation and during the phar-macokinetic study were prepared by spiking 100 µL blank whole blood with 50 µLworking solutions correspondingly.

Extraction procedure for plasma samples

A 0.1 mLvolume of whole blood sample was transferred to 2 mL RIA(Radio Immune Assay) vial and then 50 µL of the internal work so-lution (5 µg/mL methanol water 50:50v/v) was spiked. The mixture was vortexed for 30s; 0.5 mL aliquot of extraction solvent (t-butyl methyl

ether) was added using dispensette organic (Brand GmbH, Postfach, Germany).The analyte and IS were extracted from whole blood by vortexing for 10 min using heidolph vibramax 110 (Germany). Then sample was centrifuged using micro centrifuge (5415R Eppendorf) for 5min at 10000 rpm. Following centrifugation the supernatant was transferred to clean glass test tubes and then evaporated to dryness us-ing TurboVap LV Evaporator (Zymark, Hopkinton, MA, USA) at 40 ºC under a stream of nitrogen. The residues were reconstituted with 100 µL of mobile phase and aliquots of 20 µL were injected into the Chro-matographic system.

Bioanalytical method validation

Selectivity and lower limit of quantification (LLOQ): To investi-gate the selectivity of the method, rat whole blood blank samples from 6 different lots were pretreated and analyzed at LLOQ.

Calibration curve: A calibration curve was constructed from a blank sample (a whole blood processed without an IS), a zero sample (a whole blood processed with IS) and eight non-zero samples covering the total range (20.931-1000.703 ng/mL), including LLOQ. Eight sam-ples of each concentration were measured and the curves were fitted by a linear weighted (1/x2) least squares regression method through the measurement of the peak-area ratio of the analyte to IS. The calibration curve had to have a correlation coefficient (r2) of 0.999 or better. The acceptance criterion for each back-calculated standard concentration was 15% deviation from the nominal value except LLOQ, which was set at 20%.

Recovery: The extraction recoveries were determined at three con-centration levels (50.285, 478.908 and 798.179 ng/mL) for tacrolimus by comparing the analyte peak areas obtained from the quality control samples (n = 6) after extraction with those obtained from the corre-sponding unrestricted reference standards prepared at the same con-centrations. Recovery of IS was evaluated by comparing the mean peak areas of 6 extracted Blank+IS samples to mean peak areas of 6 neat ref-erence solutions (unprocessed) of the same concentration.

Precision and Accuracy: To evaluate the precision and accuracy of the method, QC samples at four concentration levels (21.120, 50.285, 478.908 and 798.179 ng/mL) were analyzed in six replicates on three validation days. The assay precision was calculated by using the relative standard deviation (RSD).The assay accuracy was expressed as relative error (RE %), i.e. (measured concentration-nominal concentration)/ (nominal concentration) ×100.

Stability: The stability of the stock solutions and working solutions of tacrolimus and IS, which were stored at 4ºC -8ºC for 15 days and at room temperature (25ºC) for 5 h, was tested by comparing the instru-ment response with that of freshly prepared solutions. The analytes were considered stable when the intensities ranged between 85% and 115% of the initial solutions. The stability of tacrolimus in rat whole blood was evaluated by analyzing replicates (n=6) of whole blood samples

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Figure 1: Chemical structures of tacrolimus (I) and internal standard Pimecro-limus (II)

Parameter ValueSource temperature (°C) 350Dwell time per transition (milli seconds) 200Gas flow/Drying Gas (L/min) 6Nebulizer gas (psi) 25Fragmentor (V) 200Collision energy (V) −10Ion spray voltage (V) −5500Mode of analysis NegativeIon transition for Tacrolimus m/z 802.4→560.2Ion transition for Pimecrolimus m/z 808.4→548.2

Table 1: Tandem mass spectrometer main working parameters.

Volume 2 • Issue 2 • 1000118J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal

Citation: Babu ARS, Thippeswamy B, Vinod AB (2011) Determination of Tacrolimus in Rat Whole Blood Utilizing Triple Quadrupole LC/MS. J Anal Bioanal Techniques 2:118. doi:10.4172/2155-9872.1000118

Page 3 of 5

that were exposed to different conditions (time and temperature) at two concentrations (50.285 and 798.179 ng/mL). These results were com-pared with those results obtained from freshly prepared whole blood samples. The analyte was considered to be stable in biological matrix and acceptance criteria is ±15%.The long term stability was determined after exposure of the spiked samples at -20°C for 10 days. The freeze thaw stability was evaluated after complete three freeze thaw cycles (-20 ºC to -25 ºC) on consecutive days.

Matrix effects: According to the method described by Matuszewski et al. [29], assessed the matrix effects (MEs), whether the potential ion suppression or enhancement owing to the co-eluting matrix compo-nents existed in the present experiment. The corresponding peak areas of tacrolimus from the spike-after-extraction samples at one concentra-tion level (50.285 ng/mL) were then compared to those of the respective unprocessed or aqueous standard. Matrix effects of IS were investigated at concentration level (5 µg/mL) in similar way.

Results and discussionLC-MS/MS Optimization

Electrospray MS–MS was used to analyze the compound tacrolim-us.Negative ionization was selected to quantify the analyte because neg-ative ion mass spectrometry gave a deprotonated molecular ion without adduct formation over positive ionization (Figure 2). The combination of chromatographic separation by HPLC and successive mass filtrations by monitoring the transition of the deprotonated ion to product ion provided excellent specificity for tacrolimus and internal standard. The negative ion electro-spray mass spectrum of the analyte and IS gave a deprotonated molecular ions at m/z 802.4, m/z 808.4 and product ions at m/z 560.2, m/z 548.6 respectively.

Thus, the MRM technique was chosen for the assay development. The MRM state file parameters were optimized to maximize the re-sponse for the analyte. The approach applied to the development of this method was based on the literature survey done on tacrolimus, which form adducts for the quantization by LC–MS/MS [30-34]. Signal inten-sities are affected by the presence of ion in the mobile phase concentra-tions. Signal intensities are also affected by the type of adduct formed.

Taylor and Johnson [33] state that the resultant product ion spectra of the sodium adduct contains more fragmentation and thus gives a less sensitive response for selected reaction monitoring. Sodium adduct of sirolimus is more stable than the ammonium adduct and thus requires more energy to produce fragmentation. So they added ammonium ac-etate to the mobile phase in order to produce ammoniated species, [M + NH4] + for mass spectrometric detection of sirolimus using selected reaction monitoring.

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Representative negative ion Triple Quadrupole LC-MS spectra ob-tained (A) Precursor ion of tacrolimus (B) product ion of tacrolimus(C) Full scan of IS and (D) product ion of IS along with precursor ion.

Tacrolimus - 8 Levels, 8 Levels Used, 8 Points, 8 Points Used, 24 QCs

Relative Concentration0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2

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Figure 3: Calibration carve for extracted tacrolimus from rat whole blood.

Nominal concentration (ng/ml) n Precision (%) Accuracy (%)

20.931 5 1.93 102.641.861 5 3.45 96.4549.835 5 0.92 99.4599.169 5 1.02 97.50240.169 5 2.17 97.60480.337 5 0.28 101.60800.703 5 1.48 100.151000.703 5 1.55 104.75

Table 2: Precision and accuracy data of back-calculated concentrations of calibration samples for tacrolimus in rat whole blood.

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Figure 4: LC−MRM ion-chromatograms resulting from the analysis of blank (drug and internal standard free) rat whole blood for tacrolimus (I) and IS (II).

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Figure 5: LC−MRM ion-chromatograms resulting from the analysis of blank (drug-free spiked with IS) rat whole blood for tacrolimus (I) and IS (II).

Figure 2:

Volume 2 • Issue 2 • 1000118J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal

Citation: Babu ARS, Thippeswamy B, Vinod AB (2011) Determination of Tacrolimus in Rat Whole Blood Utilizing Triple Quadrupole LC/MS. J Anal Bioanal Techniques 2:118. doi:10.4172/2155-9872.1000118

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But in case of Christians et al. [2] method, even if 2mM ammo-nium acetate was added to the mobile phase to induce formation of [M +NH4]+ at the expense of other ion species, [M+Na]+ still gave a significant signal. Addition of sodium ions to the mobile phase almost completely suppressed formation of other ions. So, they added sodium formate to the mobile phase for mass spectrometric detection of cyclo-sporine, tacrolimus, sirolimus and everolimus using selected ion moni-toring.Therefore, sensitivity, robustness and ruggedness of the method are questionable. There is a need of rugged method in high-throughput bioanalysis. This method is robust, simple and rapid which makes it an attractive procedure in high-throughput bioanalysis.

Selection of mobile phase and internal standard

Different mobile phases were evaluated to improve HPLC separa-tion and enhance sensitivity in MS. A binary system using a mobile phase of 90% methanol and 10% 2mM of ammonium acetate buffer was optional for the analyte with respect to peak shape and mass spectral response. Under this condition, the retention times of both analyte and IS were approximately 1.134 and 1.230 min respectively. The mobile phase used guaranteed good repeatability of retention times. The total run time for each sample was 2.5 min.

The use of an internal standard was required in the LC–MS/MS as-say for two reasons: to compensate for loses during extraction and to compensate for the variable detection sensitivity of the MS. The more samples we ran on the MS, the more the skimmers and the MS source were contaminated, and more the sensitivity decreased. Thus, Pimecro-limus good choice as the IS for tacrolimus since being an acid with pKa value, its solubility in mobile phase, its chromatographic and extrac-tion properties, which are similar to tacrolimus. It was expected to give similar recovery on Liquid-Liquid extraction and MS/MS response in the negative ion mode.

Calibration curves

Calibration curve was linear over the concentration range of 20.931 -1000.703 ng/mL for the analyte. The eight-point calibration curve gave acceptable results for the analyte and was used for all the calculations. The mean correlation coefficient of the weighted (1/x2) calibration curve generated during the validation was 0.999 for the analyte (Fig-ure 3). Table 2 summarizes the calibration curve results for the analyte. The precision and accuracy for the analyte covering the concentra-tion of 20.931 -1000.703 ng/mL ranged from 0.28 to 3.45 and 96.45 to 104.75%, respectively. The calibration curve obtained as described above was suitable for generation of acceptable data for the concentra-tions of the analyte in the samples during the validations for tacrolimus in rat whole blood.

Specificity

The specificity of the method was examined by analyzing the blank whole blood extract (Figure 4) and spiked only with the internal stan-dard (Figure 5). As shown in (Figure 4), no significant interference in the blank Whole blood traces was seen from endogenous substances in drug-free rat whole blood at the retention time of the analyte. Figure 5 shows the absence of interference from the internal standard to the MRM channels of the analyte. Figure 6 depicts a representative ion-chromatogram for the LLOQ (20.931 ng/mL) of the calibration curve. Excellent sensitivity was observed for 20 µL injection volume corre-sponding to 20.931 ng/mL on-column.

Recovery

The extraction recovery of tacrolimus was 23.92% on average and the recovery of IS was 18.52% at the concentration used in the assay (5µg/mL). Recovery of analyte and IS were low but it was consistent, precise and reproducible.

Lowest concentration

The LLOQ of tacrolimus in rat whole blood assay was 20.195 ng/mL. Although peaks were detected at the concentration of 10 ng/mL with a signal-to-noise ratio above 5, the precision and accuracy did not meet the acceptance criteria (<±20%). The between-batch precision at the LLOQ-expressed as RSD was 1.20%. The between-batch accuracy expressed as relative error (RE) was 107.98% (Table 3). The within-batch precision was 9.5% and the accuracy was 106.2% for tacrolimus.

Precision and accuracy

All the values are summarized in Table-3. The middle and upper quantitation levels of tacrolimus ranged from 50.285 to 798.179 ng/mL in rat whole blood. For the between batch experiment, the precision ranged from 1.07 to 8.01%. For the within batch experiment, the preci-sion and accuracy for the analyte met the acceptance criteria (<±15%) and precision was below 4.88% at all concentrations tested.

Freeze -thaw stability

The freeze-thaw stability of the analyte was determined by measur-ing the assay precision and accuracy for the samples which underwent three freeze-thaw cycles. The stability data were used to support repeat analysis. The frozen whole blood samples containing the analyte was thawed at room temperature for 2–3 h, refrozen for 12–24 h, thawed for 2–3 h, refrozen for 12–24 h, thawed and then analyzed. The results

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IIFigure 6: Representative LC–MRM ion-chromatograms resulting from the Analysis of 20.931 ng/ml (LLOQ) of tacrolimus (I) spiked with the IS (II,) 5µg /mL per sample).

Spiked concentration (ng/ml) n Precision (%) Accuracy (%)Freeze-thaw stability50.285 (LQC) 6 4.24 103.58798.179 (HQC) 6 2.55 105.36Long-term frozen storage stability50.285 (LQC) 6 4.72 103.96798.179(HQC) 6 2.48 103.14

Table 4: Stability of rat whole blood samples of tacrolimus.

Table 3: Precision and accuracy data from between-batch experiment for tacroli-mus in rat whole blood.

Nominal concentration (ng/ml) n Precision (%) Accuracy (%)

LLOQ (21.120) 24 1.20 107.98Low concentration (50.285) 24 1.94 100.20

Medium concentration (478.908) 24 8.01 95.47

High concentration (798.179) 24 1.07 100.18

Volume 2 • Issue 2 • 1000118J Anal Bioanal TechniquesISSN:2155-9872 JABT, an open access journal

Citation: Babu ARS, Thippeswamy B, Vinod AB (2011) Determination of Tacrolimus in Rat Whole Blood Utilizing Triple Quadrupole LC/MS. J Anal Bioanal Techniques 2:118. doi:10.4172/2155-9872.1000118

Page 5 of 5

showed that the analyte was stable in rat whole blood through three freeze-thaw cycles. The precision ranged from 2.55 to 4.24% and the accuracy ranged from 103.58 to 105.36% (Table 4). The results dem-onstrated that, rat whole blood samples could be thawed and refrozen without compromising the integrity of the samples.

Long-term storage stability

The storage time in long-term stability evaluation, the time between the first sample collection and the last sample analysis. The sample long-term storage stability at −20ºC was evaluated to establish accept-able storage conditions for Pharmacokinetic samples. Aliquots of rat whole blood samples spiked with analyte at concentrations of 50.285 and 798.179 ng/mL were analyzed on day 1. Then the samples from the same pools were analyzed against calibration curves from freshly prepared standards after storage at −20ºC for 10 days. The precision and accuracy for the analyte on day 10 ranged from 2.48 to 4.72 and 103.14 to 103.96%, respectively

ConclusionsIn summary, tacrolimus is quantified in rat whole blood by Triple

Quadrupole LCMS negative ionization mode without adduct forma-tion using MRM.The method described is simple, rapid, sensitive, specific and fully validated as per FDA guidelines. The cost effective-ness, simplicity and speed of liquid-liquid extraction and sample total run time of 2.5 mins per sample make it an attractive procedure. The validated method allows quantization of tacrolimus in the 20.931 to 1000.703 ng/mL range.

Acknowledgements

Authors wish to acknowledge the support received from Mr.Balaji Karthikeyan, Vice President, G7Synergon Pvt Ltd, Tatanagar, Bangalore. India.

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