MS method for tacrolimus determination in human whole blood samples – A compensation of matrix effects

Accepted Manuscript Isotope–labeled versus analog internal standard in LC–MS/MS method for tacrolimus determination in human whole blood samples – A compensation of matrix effects

Magdalena Bodnar-Broniarczyk, Tomasz Pawiński, Paweł K. Kunicki PII: DOI: Reference:

S1570-0232(18)31247-9 https://doi.org/10.1016/j.jchromb.2018.11.026 CHROMB 21447

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

14 August 2018 25 October 2018 27 November 2018

Please cite this article as: Magdalena Bodnar-Broniarczyk, Tomasz Pawiński, Paweł K. Kunicki , Isotope–labeled versus analog internal standard in LC–MS/MS method for tacrolimus determination in human whole blood samples – A compensation of matrix effects. Chromb (2018), https://doi.org/10.1016/j.jchromb.2018.11.026

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ACCEPTED MANUSCRIPT Isotope–labeled versus analog internal standard in LC-MS/MS method for tacrolimus determination in human whole blood samples – a compensation of matrix effects.

Magdalena Bodnar-Broniarczyka, Tomasz Pawińskia, Paweł K. Kunickia, b, *. Department of Drug Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha

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a

Clinical Pharmacology Unit, Department of Medical Biology (previous name: Department of

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b

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1, 02-097 Warsaw, Poland.

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Clinical Biochemistry), Institute of Cardiology, Alpejska 42, 04-628 Warsaw, Poland.

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E-mail addresses:

[email protected] (MB-B), [email protected] (TP),

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Conflict of interest:

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[email protected] (PKK).

The authors declare no conflict of interest regarding the content of this article.

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Funding source:

Warsaw.

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This study was supported in part by Grant No. FW-22/N/2015 of the Medical University of

*Corresponding author: Paweł K. Kunicki, PhD, Department of Drug Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland. E-mail: [email protected]. ORCID: 0000-0002-4552-0021

ACCEPTED MANUSCRIPT Abstract The aim of this work was to develop and to validate LC-MS/MS method for tacrolimus (TAC) determination in whole blood samples using two different types of internal standards (IS): an isotope-labeled TAC13C,D2 and a structural analog ascomycin (ASC). Matrix effects (ME) were evaluated to determine their influence on validation parameters.

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The LC-MS/MS analyses were performed using a 4000 QTRAP® mass spectrometer

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(AB Sciex) coupled to a HPLC 1260 Infinity system (Agilent Technologies). The [M+NH4]+ adducts were monitored with mass transitions of: 821.5→768.4 m/z for TAC, 809.5→756.4

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m/z for ASC, and 824.6→771.5 m/z for TAC13C,D2. Blood samples were treated with 0.1 mol/L

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zinc sulfate - acetonitrile (50:50, v/v) then extracted with tert-butyl methyl ether. ME evaluations were performed by preextraction addition (n=6), postextraction addition (n=8), and

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repeated measures (n=8) of reference solutions, separately for both ISs. ME, absolute recovery (AR) and process efficiency (PE) were calculated. Low (1.5 ng/mL) and high (16 ng/mL) TAC

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concentrations were tested.

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The method was successfully calibrated in a range of: 0.5-20 ng/mL. Both ISs provided satisfactory imprecision (<3.09% and <3.63% for TAC13C,D2 and ASC, respectively) and

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accuracy (99.55-100.63% and 97.35-101.71% for TAC13C,D2 and ASC, respectively). Similar ARs were found for all three compounds yielding: 74.89-76.36% for TAC, 78.37% for

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TAC13C,D2 and 75.66% for ASC. Significant ME were observed yielding on the average: 16.04% and -29.07% for TAC, -16.64% for TAC13C,D2 and -28.41% for ASC. Consequently, PE was 64.11% and 53.12% for TAC, 65.35% for TAC13C,D2 and 54.18% for ASC. ME for TAC were perfectly compensated (TAC/IS ratio) in each sample resulting in mean percent value of: 0.89% and -0.97% for TAC13C,D2 and ASC, respectively. An IS ascomycin presented a performance equivalent to TAC13C,D2 in LC-MS/MS method developed for TAC monitoring.

ACCEPTED MANUSCRIPT Keywords tacrolimus, LC-MS/MS, internal standard, matrix effects, validation

1. Introduction Tacrolimus (TAC) is a primary immunosuppressive drug administered in transplant

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patients for protecting against both acute and chronic rejection [1]. Currently, majority of

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patients after organ transplantation receive TAC as a leading agent in one of many different

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immunosuppressive protocols. Safety and efficacy of TAC have been proved in randomised clinical trials [2]. TAC is characterised by a narrow therapeutic range, as well as by high inter-

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and intra-subject pharmacokinetic variability, thus monitoring TAC concentration in blood samples is mandatory for proper dose individualisation [1-6]. Therapeutic range for TAC

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steady-state trough concentration is most often defined between 5 and 20 ng/mL, however lowdose schemes have been recently introduced with measured TAC concentrations starting from

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2-3 ng/mL [2, 7]. Medical laboratories performing TAC therapeutic drug monitoring (TDM)

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benefit both from immunoassays and chromatography [7-11]. The chosen analytical technique is, or rather should be, dedicated to performance of a specific laboratory, number of samples to

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be analyzed and funds available for spending in TDM. Ready-to-use immunoassays are preferable for small or transplant oriented medical laboratories. Currently, there are five major

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immunoassays utilized in TDM laboratories for TAC whole blood monitoring: EMIT, ACMIA, ECLIA, QMS and the most popular CMIA [11]. However, a primary disadvantage of immunoassay in comparison with chromatography is its reliability downgraded by problems related to specificity issues. Chromatography enables simultaneous measuring parent drug and its metabolite(s). Properly validated and maintained chromatographic method guarantees adequate, specific determinations free from most of interferences [7, 12]. Mass spectrometry is a detection of choice for TAC and currently liquid chromatography with tandem mass

ACCEPTED MANUSCRIPT spectrometry (LC-MS/MS) is generally accepted as a reference technique characterized by good selectivity, adequate range and relatively short time of analysis [3, 10, 13-16]. TAC is extensively bound to erythrocytes with blood to plasma concentration ratio of 15:1, what implies whole blood but not plasma as the appropriate matrix for TAC determination [4, 5]. Prior to chromatography, solid phase extraction (SPE) [12, 17-22], liquid-liquid extraction

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(LLE) [23-27] or on-line sample clean-up [27] may be used for sample pretreatment. Internal

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standard (IS) is required for proper method calibration. Taylor et al. evaluating different ISs for

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another immunosuppressant cyclosporine, differentiated 3 types of IS: an isotope-labeled IS (ILIS), a structural analog IS (AIS) and a structurally unrelated IS (URIS) [28]. Reviewing LC-

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MS/MS methods published for TAC monitoring, we found several compounds reported as ISs: deuterated TAC (TAC13C,D2) [22, 29], 32-O-acetyloTAC [30], ascomycin (ASC) [3, 12, 18,

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19, 26, 31, 32], sirolimus [21], and tamsulosin [23]. The close similarity of chemical structures between analyzed drug and applied IS is critical for successful compensation of matrix effects

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(ME) thus ILIS is recognized as a first choice compound [2, 8, 10, 13, 15, 22, 33-36]. Generally,

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there is a consensus between professionals that ILIS is always better than AIS which, in turn, takes advantage over URIS [6, 28, 36, 37]. Despite mentioned reasons, an analyst often decides

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to use AIS or even URIS due to lower cost and/or easier availability. However, this overall attractiveness may be easily lost by ME, which is a weakness characteristic for LC-MS/MS,

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especially if using electrospray ionization (ESI) [22, 36-38]. Exactly ESI with positive natrium or ammonium adducts was a common ionisation mode in several TAC methods [3, 12, 17, 25, 29, 31]. Looking for the best IS, ascomycin is still recognized as an attractive alternative for deuterated TAC, therefore it seems to be reasonable to directly compare analytical utility of these two ISs in LC-MS/MS method intended for routine TAC determination. A comprehensively prepared study has been recently presented by Valbuena et al. [29]. The authors evaluated four primary immunosuppressive drugs including TAC for comparing the

ACCEPTED MANUSCRIPT effect of isotopically labeled or structural analog ISs on the analytical method performance. They reported high similarity of validation results obtained with both types of IS, however it should be emphasised that no ME were observed concomitantly. Therefore, the paper by Valbuena et al. did not verify a case when significant ME is observed during method validation, which we may have expected from preliminary experiments preparing our procedure for TAC

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[29].

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The aim of the work was to develop and to validate LC-MS/MS method for TAC

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determination in human whole blood samples using two different ISs for quantification purposes. A special interest was dedicated to comparison of ILIS and AIS type compounds for

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method performance. Finally, ME were evaluated to determine their influence on validation

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parameters.

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2.1. Chemicals and reagents

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2. Materials and methods

Tacrolimus (≥98% chemical purity) was a gift from Astellas Pharma Inc. (Tokyo, Japan); stable

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isotope-labeled tacrolimus (TAC13C,D2) (88,2% chemical purity, 86,0% isotopic purity) were purchased from Toronto Research Chemicals Inc. (Toronto, ON, Canada), while ascomycin

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(ASC) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The chemical structures of TAC, ASC and TAC13C,D2 are presented in Figure 1. The substances were stored at -20oC. The reagents for chromatography: hyper LC/MS-grade methanol and water as well as zinc sulfate heptahydrate were purchased from Merck Millipore (Darmstadt, Germany). Hyper LC/MSgrade acetonitrile, formic acid and HPLC-grade tert-butyl methyl ether, were from J.T. Baker (Deventer, The Netherlands), whereas ammonium acetate Optigrade was from LGC Standards

ACCEPTED MANUSCRIPT (Wesel, Germany). Reagent-grade deionized water was produced using a Millipore SimPak®1, Simplicity 185 (Millipore, France).

2.2. Equipment and chromatographic conditions LC-MS/MS technique was selected for TAC determination. Analyses were performed using

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4000 QTRAP® triple quadrupole mass spectrometer (AB Sciex, Concord, ON, Canada),

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coupled to HPLC Agilent 1260 Infinity liquid chromatography system (Agilent Technologies,

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Palo Alto, CA, USA) consisting of: binary pump (G1312B), degasser (G4225A), thermostated column compartment (G1316A), autosampler (G1367E), thermostat for the autosampler

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(G1330B). The analytical column used was Poroshell 120 EC-C18 (4.6 x 50 mm, 2.7 µm), which was maintained at 50ºC and guarded with Poroshell 120 EC-C18 (4.6 x 5 mm, 2.7 μm)

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precolumn, both from Agilent Technologies. The mobile phase pumped in a gradient mode consisted of (A): ammonium acetate (2.5 mmol/L) and 0.1% formic acid in water and (B):

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ammonium acetate (2.5 mmol/L) and 0.1% formic acid in methanol. A binary step gradient at

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a flow rate of 0.75 mL/min was employed. The gradient program was as follows: 90% of solution A and 10% of solution B from the start of analysis till 2.0 min, then changed to 5% of

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solution A and 95% of solution B from 2.0 min to 6.0 min. At 6.1 min the mobile phase was reverted back to 90% of solution A and 10% of solution B (as was in the initial phase). The

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total time of instrumental analysis was 8 min. The autosampler temperature was maintained at 4°C and the injection volume was set at 10 μL. Analyst 1.6.1 software (AB Sciex) was used for peak area counting, calibration fitting, TAC concentrations calculating and also signal-to-noise ratio determining.

2.3. Mass spectrometry

ACCEPTED MANUSCRIPT Multiple reaction monitoring (MRM) was performed using electrospray ionisation (ESI) in positive-ion mode. The ammonium adduct of each analyte [M+NH4]+ was monitored with mass transitions of: 821.5→768.4 m/z, 809.5→756.4 m/z and 824.6→771.5 m/z for TAC, ASC and TAC13C,D2, respectively. A dwell time of 150 ms was used for each mass transition. Nitrogen was used as both the curtain and collision gas. The compound-specific parameters were set to

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the following values: a collision energy of 31 V, an entrance potential of 10 V and the

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declustering potentials of 96 V for TAC and 91 V for both ISs used. Ion source parameters were

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as follows: an ESI voltage of 4.5 kV, a desolvation temperature of 400°C (to achieve optimal

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signal-to-noise ratio), the curtain gas of 20, GS1 of 50 and GS2 of 60 units.

2.4. Stock and working solutions

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Stock solutions of TAC, ASC and TAC13C,D2 (100 μg/mL) were prepared in methanol using weighed amounts and stored at −20°C. The second stage stock solutions (1 μg/mL) of all three

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compounds were prepared in methanol from primary stock solutions and stored at −20°C. The

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working solutions (25 ng/mL) were prepared from the second stage stock solutions by adequate

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dilution in methanol/water 50/50 (v/v) and further stored at 4°C.

2.5. Calibrators and quality controls

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The calibration standards (CS) were prepared by spiking blank blood with TAC working solution to obtain drug concentrations of: 0.5, 1.0, 3.0, 5.0, 7.0, 10.0, 15.0 and 20.0 ng/mL. The quality control (QC) samples were prepared in same way at three levels: 0.7 ng/mL (lower LQC), 1.5 ng/mL (medium - MQC), and 16.0 ng/mL (higher - HQC). During calibration, a blank sample spiked only with internal standard was analyzed following standard procedure.

2.6. Sample preparation

ACCEPTED MANUSCRIPT The drug-free whole blood used in this study was obtained from Regional Blood Donation and Blood Treatment Center in Warsaw, Poland. An aliquot of 0.25 mL whole blood sample (fortified accordingly with TAC in case of CS or QC samples) was spiked with 2.5 ng (100 µL) or 3.75 ng (150 µL) of IS working solution (ASC and TAC-13C,D2, respectively) next vortexed and allowed to stand. To perform the protein precipitation, the sample prepared as described

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above, was treated with 1.0 mL of 0.1 mol/L zinc sulfate - acetonitrile (50:50, v/v). This mixture

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was then vigorously rotary mixed for 20 minutes at room temperature. After centrifugation

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(3000 rpm for 10 min at 5°C) the supernatant was quantitatively transferred into a glass tube and next the extraction with 3.0 mL tert-butylmethyl ether using a rotary mixer (30 min at 1200

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rpm) was performed. Subsequently, sample centrifugation at 3000 rpm for 10 min at 5°C was done, after that the organic layer was transferred into another glass tube and evaporated to

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dryness under a stream of nitrogen in a thermostatically controlled water bath maintained at 50°C. The dried extract was reconstituted in 500 µL of the mobile phase (methanol/water, 50/50

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v/v), it was subsequently transferred into an autosampler vial and finally 10 µL aliquot was

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2.7. Method validation

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injected into the chromatographic system.

The validation of the method was performed according to European Medicines Agency (EMA)

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guideline [39]. Validation parameters ie. selectivity, accuracy and precision, range with lower limit of quantification (LLOQ), carry-over, calibration and linearity were evaluated independently for both ISs. A special caution was given to stability examination and to tests evaluating ME.

2.7.1. Stability 2.7.1.1. Autosampler stability

ACCEPTED MANUSCRIPT Autosampler stability was tested using four replicates for each TAC concentration level (low QC ~1.5 ng/mL and high QC ~16 ng/mL). Samples were measured immediately after preparation (initial 0 h) and subsequently after 4, 8, 12 and 24 h of sample storage in the autosampler chamber maintained at 4°C.

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2.7.1.2. Short-term stability

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Short-term stability experiment was performed at low QC (~1.5 ng/mL) and high QC (~16

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ng/mL) TAC concentration levels. Spiked TAC samples were evaluated immediately after preparation according to the procedure described in Section 2.6 (standard analytical procedure,

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n=4). Another set of samples for each concentration level was prepared accordingly and after 4 h of resting at ambient temperature the preparation procedure was continued (stability before

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preparation procedure, n=4). Simultaneously, the next set of samples was prepared according to the procedure until it was interrupted for 4 h of resting at ambient temperature just after

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evaporation step (see Section 2.6) (stability after preparation procedure, n=4).

2.7.1.3. Long-term stability

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The long-term stability, similarly to other experiments was tested using both low QC (~1.5 ng/mL) and high QC (~16 ng/mL) TAC samples in four replicates (n=4). Prepared samples

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were analyzed immediately after preparation and then stored in a refrigerator at 4°C. After 1, 3, 4, 9, 11, 13 and 15 weeks, the samples were carefully mixed and subsequently analyzed when reaching ambient temperature.

2.7.1.4. Working solution stability

ACCEPTED MANUSCRIPT The solutions of TAC, TAC13C,D2 and ASC (25 ng/mL in methanol/water 50/50) were initially analyzed in triplicate: immediately after preparation, than in three occasions when stored refrigerated at 4°C during 4-week observation.

2.7.2. Matrix effect

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Two independent methods, postcolumn infusion and postextraction addition were evaluated for

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qualitative and quantitative ME testing, respectively [40]. The postcolumn infusion was

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conducted by infusing TAC or TAC13C,D2 or ASC (100 ng/mL) postcolumn (0.5 mL/min) and injecting sample under the established chromatographic conditions. Postextraction addition

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technique is accepted by the EMA guideline [39]. A comprehensive quantitative approach included ME evaluations using 6 different blood samples for preextraction addition, 8 different

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blood samples for postextraction addition, and repeated measures (n=8) of reference solutions in each experiment, separately for both ISs. Aiming to avoid any potential interference between

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alternative TAC13C,D2 and ASC, the ME experiments were performed independently for both

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ISs. Blood samples were obtained from transplant patients not treated with TAC. ME, absolute recovery (AR) and process efficiency (PE) were calculated accordingly to Taylor [41].

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were measured.

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Similarly to stability testing, both low (1.5 ng/mL) and high (16 ng/mL) TAC concentrations

2.8. Clinical application Supplementary to validation experiments, the comparison using 35 spiked and 18 pooled samples from Tacrolimus International Proficiency Testing Scheme (IPTS, Analytical Services International Ltd) was performed as well with two ISs tested. The results of TAC determinations obtained using TAC13C,D2 or ASC in IPTS samples, were statistically evaluated

ACCEPTED MANUSCRIPT by means of linear regression model, Passing-Bablok regression and Bland–Altman procedure adequate for estimating bias all available from statistical software MedCalc®.

3. Results 3.1. Method development

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TAC determination in human whole blood samples was relied on LC-MS/MS technique. An

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Poroshell 120 EC-C18 was chosen as optimal column for chromatographic separation with

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simple mobile phase consisted of two solutions (ammonium acetate plus formic acid in water and in methanol) operated in a gradient mode. Chromatographic separation as well as MS

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parameters were adjusted experimentally. Taking into consideration low TAC concentration expected in patients’ samples (low-dosing immunosuppressive protocols) we have paid more

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attention to satisfactory validation parameters like LLOQ and precision, than to challenging method simplicity or shortening time of analysis. As the LC-MS/MS equipment in our

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laboratory was simultaneously utilized for other research analyses, we tried to develop a

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procedure resulting in chromatographically clean sample. Protein precipitation followed by liquid-liquid extraction was thus assumed. Simple precipitation with acetonitrile with

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subsequent extraction with tert-butylmethyl ether resulted in recovery between 68-74%, however the sample was not clean enough. Later, 0.1 mol/L zinc sulfate - acetonitrile (50:50,

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v/v) was used instead acetonitrile improving recovery (up to 75-80%) as well as sample clarity, what finally fulfilled our requirements. In described (Sections: 2.2, 2.3 and 2.6) analytical conditions the compounds of interest were eluted in retention time of: 3.0-3.1 min for each of determined analytes i.e. TAC, TAC13C,D2 and ASC, presenting no interference in chromatograms recorded. Total run-time was 8 min.

3.2. Method validation

ACCEPTED MANUSCRIPT 3.2.1. Selectivity Accordingly to EMA guideline [39], six different sources of whole blood obtained from Regional Blood Donation and Blood Treatment Center (not containing TAC) were analyzed showing no chromatographic interference. The signals from endogenous or unknown substances were less than 20% of LLOQ value (i.e. <0.1ng/mL) for TAC; and were less than

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5% for ISs fulfilling EMA requirements.

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3.2.2. Carry-over effect

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Carry-over effect was evaluated by determining blank sample immediately after the highest TAC calibrator sample (20 ng/mL) in established chromatographic conditions. The data were

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collected from 20 sets of different analytical runs, separately for ASC and TAC13C,D2. Carryover effect for TAC was calculated in percentage at 1.41 ± 1.15% and 1.88 ± 1.49% (n=19) of

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the signal recorded at the same run for LLOQ sample. Carry over for ISs were found at a percentage level of: 0.04 ± 0.03% and 0.30 ± 0.22% (n=19) for ASC and TAC13C,D2,

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respectively. As the signals were well below EMA acceptance limits (<20% of LLOQ for the

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analyte / <5% for the IS) [39] no carry-effect was concluded. 3.2.3. Calibration and linearity

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The linearity of the method was evaluated for TAC blood concentration in the range between 0.5 and 20 ng/mL from a set of 25 calibration curves. Each curve consisted of standards at 8

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levels, a blank sample and a zero sample. A weighted (1/x) linear regression was used in constructing the calibration curve to ensure optimal fitting at low TAC concentrations. Calibration lines were characterized by satisfying values of a coefficient of determination: r2=0.9994 ± 0.0004, and r2=0.9987 ± 0.0035 for ASC and TAC13C,D2, respectively. The backcalculated TAC concentrations were far inside the limits (±15% of the nominal value, ±20% for LLOQ) for inaccuracy mean yielding: <0.61% and < 2.55% for ASC and TAC13C,D2, respectively.

ACCEPTED MANUSCRIPT 3.2.4. LLOQ LLOQ value was experimentally set at 0.5 ng/mL. Required level of accuracy and precision was easily obtained for both ISs used. The accuracy was 99.55% / 100.20% and 100.99% / 97.35% for TAC13C,D2 and ASC, respectively. The corresponding imprecision was calculated as: 1.08% / 3.09% and 1.39% / 3.63% for TAC13C,D2 and ASC, respectively. Detailed

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information is presented in Table 1.

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3.2.5. Accuracy and precision

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The accuracy and precision were evaluated both in within-run as well as between-run experiments. Three QC levels (1.5, 7 and 16 ng/mL) and LLOQ (0.5 ng/mL) were included.

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The results are showed in Table 1.

The within-run accuracy was measured 99.55-100.48% and 100.08-101.59% for TAC13C,D2

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and ASC, respectively, while between-run accuracy was measured 99.95-100.63% and 97.35101.71% for TAC13C,D2 and ASC, respectively.

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The within-run imprecision was calculated at 0.78-1.08% and 0.72-1.39% for TAC13C,D2 and

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ASC, respectively, while between-run imprecision was found 1.21-3.09% and 0.77-3.63% for TAC13C,D2 and ASC, respectively.

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Obtained results easily fit the general analytical requirements from EMA (accuracy within 85115%, imprecision less 15%) but also recently recommended for TDM dedicated assays,

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achievement of imprecision of at least ≤10% [7, 39].

3.3. Stability 3.3.1. Autosampler stability The results of autosampler stability are presented in Table 2. No significant change in TAC concentration was noted during observation period. The stability of low QC was found at

ACCEPTED MANUSCRIPT 97.98% (TAC13C,D2) or 97.04% (ASC) while that of high QC was amounted to 99.69% (TAC13C,D2) or 100.40% (ASC) of the initial concentration after 24h of storage. 3.3.2. Short-term stability The results for short-term stability are presented in Table 3. Standard analytical procedure interruption for 4 hours neither before nor after sample preparation step influenced the result of

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TAC assay.

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3.3.3. Long-term stability

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The long-term stability data are included in Table 4. The results from 15-week observation proved practically no time influence on TAC concentration in described storage conditions. The

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stability of low QC was found at 100.56% (TAC13C,D2) or 99.23% (ASC) while that of high QC was found at 99.92% (TAC13C,D2) or 98.35% (ASC).

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3.3.4. Working solution stability

TAC, TAC13C,D2 and ASC working solutions (25 ng/mL) were found very stable during

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observation period lasting 4 weeks. Numerical data are presented in Table 5.

3.4. Matrix effect

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The postcolumn infusion experiments indicated the possibility of ME in described analytical conditions. From the list of tested analytes i.e. TAC, TAC13C,D2and ASC, the latter compound

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seemed to be more susceptible to suffer from ME. Quantitative evaluation is presented in detail in Table 6. No substantial influence of TAC concentration (low vs. high) tested was noted. The absolute recovery (AR) revealed similar percentage for each of three tested compounds yielding: 74.89% and 76.36% for TAC, 78.37% for TAC13C,D2 and 75.66% for ASC. However, significant ME were observed yielding on the average: -16.04% (1st test) and -29.07% (2nd test) for TAC, -16.64% for TAC13C,D2 and 28.41% for ASC. Consequently, process efficiency (PE) was 64.11% and 53.12% for TAC,

ACCEPTED MANUSCRIPT 65.35% for TAC13C,D2 and 54.18% for ASC. ME for TAC were perfectly compensated (TAC/IS ratio) in each tested sample resulting in mean percent value of: 0.89% and -0.97% for TAC13C,D2 and ASC, respectively.

3.5. Clinical application

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For the number of 53 samples covering TAC therapeutic range, drug concentrations measured

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using ASC ranged from 2.09 to 21.07, mean: 8.84 ± 4.44 ng/mL whereas TAC concentrations measured using TAC13C,D2 ranged from 2.06 to 20.73, mean: 8.86 ± 4.39 ng/mL. Regression

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analysis presented high (r2=0.9933, p<0.001) correlation between the two ISs used, similarly

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Passing-Bablok regression (Figure 2) was: y = 1.0035 x - 0.028 (95% CI for slope: 0.9867; 1.0187 and for intercept: -0.1341; +0.0967). The Bland-Altman analysis revealed a mean bias

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of -0.020 ng/mL (95% CI: -0.121; +0.081) comprising -0.78% (95% CI: -2.72; +1.16) (Figure 3). Statistical analyses confirmed no differences for TAC concentrations expected in clinical

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samples related to IS used for measurement calculation.

4. Discussion

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We presented the LC-MS/MS method for TAC determination in human whole blood samples. Developing the method, we primarily aimed for accuracy and precision, not challenging tight

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targets for sample volume, procedure simplicity or analysis time. The parameters obtained in course of validation were satisfactory for application of the method to pharmacokinetic studies, as well as for TDM. Using 0.25 mL of blood sample, LLOQ of 0.5 ng/mL was reached with a very low inaccuracy (≤2.65% and ≤0.45% for ASC and TAC13C,D2, respectively) and imprecision (≤3.63%and ≤3.09% for ASC and TAC13C,D2, respectively). Overall imprecision and inaccuracy were also well inside EMA regulations for bioanalytical methods, limiting borders for these parameters at 15% [39], and similarly in agreement with recent IATDMCT

ACCEPTED MANUSCRIPT recommendations for immunosuppressive drugs, for whom a CV ≤10% or even ≤6% for between-day imprecision has been postulated [7]. As we aimed for this publication, two different ISs were evaluated. In our method, the use of ASC (an AIS class compound) resulted in similar values of validation parameters as obtained when TAC13C,D2 (an ILIS class compound) was tested. These findings were strongly supported

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by an excellent correlation found for quality control (pooled and spiked) samples taken from

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proficiency testing scheme (IPTS). Therefore, it can be assumed that both substances may be

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equivalently accepted for TAC determination procedure that has been developed in our laboratory for application to patient samples.

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It is commonly expected that the usage of ILIS for LC-MS/MS leads to obtain better validation characteristics, finally resulting in more reliable measurements [2, 8, 10, 13, 15, 22,33-36]. As

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ILIS differs from the analyte by stable isotopic replacement for few (more than three) atoms in analyte molecule only, it demonstrates practically the same physical and chemical properties as

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the target compound. That means ILIS and the analyte show the same behaviour during whole

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analytical procedure, including a detection. Therefore, ILIS is strongly advised as the first choice compound to serve as IS [2, 8, 10, 13, 15, 22,33-36, 42]. One of the challenging issues

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in MS detection is ME, a phenomenon related to ion enhancement or (more often) ion suppression. ME is caused by constituents present in matrix, that may result in indeed great

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differences in signal measured by MS, between various patient samples, calibrators or QCs. Accordingly to FDA and EMA guidelines, as well as following scientific authorities, ME verification is currently mandatory for bioanalytical methods accomplished by MS detection [2, 7, 36, 37, 39]. If a method is affected by ME, the implications of this phenomenon for reliability of measurement can be diminished or even cancelled by use of IS. ME compensation is certainly the most expected benefit from ILIS, which in general is recognized as better than AIS. For

ACCEPTED MANUSCRIPT instance, O'Halloran and Ilett reported more accurate sirolimus measures with less imprecision when using ILIS (sirolimus-D3) vs. AIS (desmethoxyrapamycin) [43]. Similarly, better method performance and more close agreement to the results from the reference laboratory were found by Korecka et al. when they compared ILIS ([13C2D4]RAD001) vs. AIS (SDZ RAD 223-756) for everolimus determination [33].

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Conversely, a lack of significant ME is a factor enhancing successful performance of the

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method based on AIS. Such a case was presented in the publication of Valbuena et al. in which the effect of ILIS or AIS on a LC-MS/MS method for immunosuppressive agents (including

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TAC) was evaluated [29]. The compounds used for TAC determination were: TAC13C,D2 and

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ASC as ILIS and AIS, respectively. The authors concluded that AIS may be of equal value to ILIS, particularly if the method is not significantly burden by ME [29]. The question arises

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what is the value of AIS if a method for TAC suffered from ME? The data describing direct comparison of ILIS and AIS is scarce. Buchwald et al. [22] validating a method for

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immunosuppressants with ILISs found cyclosporine D (AIS) unacceptable as a result of ME

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comparing to cyclosporine-D4 (ILIS) for cyclosporine assay. The authors assumed that comparable findings could be obtained for ASC and TAC13C,D2, thus they decided to stop using

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ASC for TAC determination [22]. Contrary, Meinitzer et al. resigned fromTAC13C,D2 instead of ASC because of disproportionally higher CV values found for TAC [44]. Finally, an

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interesting impact of ILISs vs. AIS on a performance of the LC-MS/MS method for four immunosuppressive drugs including TAC was presented in abstract form by Sim et al. [45]. Beneficial reductions of imprecision after ILIS implementation to the assay were found for each drug except TAC. The authors reported that ASC did not demonstrate the same ionisation effects as the target analytes and therefore was not inferior to ILIS [45]. The experiments performed by us proved close enough values for absolute recovery of developed procedure yielding: 75-78% for each of tested compounds. Despite of a significant

ACCEPTED MANUSCRIPT ME (between -29% and -16%) found for all three analyzed compounds i.e. TAC, TAC13C,D2 and ASC it is crucial to notice that ME for TAC was perfectly compensated (TAC/IS ratio) in each tested sample. Since proportional TAC and IS measurements have been observed in literally each sample, the loss of signal proved to be negligible for method validation, what was finally confirmed by excellent accuracy and precision, regardless of internal standard used. Our

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observations applying to ASC potential in neutralisation of ME in TAC LC-MS/MS methods

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were in good agreement with other researchers. Vethe et al. reported that cyclosporine,

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everolimus and TAC were influenced by ME correspondingly as their ISs, no effects were therefore observed with respect to their calculated concentrations, that means the corrective

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capacity of IS plays a crucial role for method performance [46]. Tron et al. describing the method for TAC determination in human bile observed a relative ME close to 20%, which was

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totally corrected by ASC with mean corrected matrix factors close to 1 [47]. Analogical observations were published by Taylor et al. [3], and by Saitman et al. [32]. The important role

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of ASC for TAC LC-MS/MS methods was pointed out in a recent review on laboratory

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practices, in statement that 62% of the laboratories still use ASC as IS for TAC analysis [8]. Finally, ASC could be described as a successful exception in a perfect world of ILIS [16].

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Therefore, AIS may be equivalent to ILIS not only in case when ME is negligible [29], but also if a method has serious ME [46, 47]. The choice of the optimal IS is recognized as one of the

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most critical steps in designing a LC-MS/MS method [10, 48] and it should always be considered for particular application at particular laboratory. We fully agree to treat ILIS as a first choice compound, however, IS is only a single component in an analytical procedure and the analyst should always remember that other reagents, materials, instrumentation and local conditions have to be taken into account [6].

ACCEPTED MANUSCRIPT 5. Conclusion Ascomycin applied as internal standard, presented a performance equivalent to TAC13C,D2 in LC-MS/MS method developed for TAC monitoring. Matrix effects were successfully compensated by both internal standards justifying method application to patient samples.

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Acknowledgements The authors wish to thank Ryszard Marszałek and Agnieszka Kalicka for their technical

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assistance in TAC measurements.

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[29] H. Valbuena, M. Shipkova, S.M. Kliesch, S. Müller, E. Wieland, Comparing the effect of isotopically labeled or structural analog internal standards on the performance of a LC-MS/MS method to determine ciclosporin A, everolimus, sirolimus and tacrolimus in whole blood, Clin. Chem. Lab. Med., 54 (2016) 437–446.

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[30] A.K. Gonschior, U. Christians, M. Winkler, H.M. Schiebel, A. Linck, K.F. Sewing, Simplified high-performance liquid chromatography mass spectrometry assay for measurement of tacrolimus and its metabolites and cross-validation with microparticle enzyme immunoassay, Ther. Drug Monit., 17 (1995) 504-510. [31] J.S. Park, H.R. Cho, M.J. Kang, Y.S. Choi, A rapid and sensitive method to determine tacrolimus in rat whole blood using liquid–liquid extraction with mild temperature ultrasonication and LC–MS/MS, Arch. Pharm. Res., 39 (2016) 73–82. [32] A. Saitman, I.G. Metushi, D.S. Mason, R.L. Fitzgerald, Evaluation of the Waters MassTrak LC–MS/MS assay for tacrolimus and a comparison to the Abbott Architect immunoassay, Ther. Drug Monit., 38 (2016) 300–304.

ACCEPTED MANUSCRIPT [33] M. Korecka, R. Patel, L.M. Shaw, Evaluation of performance of new, isotopically labeled internal standard ([13c2d4]RAD001) for everolimus using a novel high-performance liquid chromatography tandem mass spectrometry method, Ther. Drug. Monit., 33 (2011) 460-463. [34] P.J. Taylor, Internal standard selection for immunosuppressant drugs measured by highperformance liquid chromatography tandem mass spectrometry, Ther. Drug. Monit., 29 (2007) 131-132.

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[35] M. Vogeser, Instrument-specific matrix effects of calibration materials in the LC-MS/MS analysis of tacrolimus, Clin. Chem. 54 (2008) 1406-1408.

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[36] A. van Eeckhaut, K. Lanckmans, S. Sarre, I. Smolders, Y. Michotte, Validation of bioanalytical LC-MS/MS assays: evaluation of matrix effects, J.Chromatogr. B 877 (2009) 2198-2207.

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[37] R. George, A. Haywood, S. Khan, M. Radovanovic, J. Simmonds, R. Norris, Enhancement and suppression of ionization in drug analysis using HPLC-MS/MS in support of therapeutic drug monitoring: A review of current knowledge of its minimization and assessment, Ther. Drug Monit., 40 (2018) 1-8. [38] T.M. Annesley, Ion suppression in mass spectrometry, Clin. Chem. 49 (2003) 1041-1044.

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[39] Guideline on bioanalytical method validation,EMEA/CHMP/EWP/192217/2009 Rev.1 Corr.2. European Medicines Agency,2011.

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[40] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS, Anal. Chem. 75 (2003) 3019-3030. [41] P.J. Taylor, Matrix effects: The Achilles heel of quantitative high performance liquid chromatography-electrospray-tandem mass spectrometry. Clin. Biochem., 38(2005) 328–334.

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[42] E. Stokvis, H. Rosing, J.H. Beijnen, Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not?, Rapid Commun. Mass. Spectrom. 19 (2005) 401-407.

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[43] S. O'Halloran, K.F. Ilett, Evaluation of a deuterium-labeled internal standard for the measurement of sirolimus by high-throughput HPLC electrospray ionization tandem mass spectrometry, Clin. Chem. 54 (2008) 1386-1389. [44] A. Meinitzer, G. Gartner, S. Pilz, M. Stettin, Ultra fast liquid chromatography-tandem mass spectrometry routine method for simultaneous determination of cyclosporin A, tacrolimus, sirolimus, and everolimus in whole blood using deuterated internal standards for cyclosporin A and everolimus, Ther. Drug. Monit., 32 (2010) 61-66. [45] A. Sim, K. Whenan, M.K. Chan, R. Thomas, T. Novos, P. Stathakis, C. Salonikas, M. Wright, R. Horvath, The importance of stable isotope internal standards for the LC-MS/MS analysis of immunosuppressant drugs in whole blood, 52nd Annual Scientific Conference of Australasian Association of Clinical Biochemists, Adelaide, 27-29.10.2014.

ACCEPTED MANUSCRIPT [46] N.T. Vethe, L.C. Gjerdalen, S. Bergan, Determination of cyclosporine, tacrolimus, sirolimus and everolimus by liquid chromatography coupled to electrospray ionization and tandem mass spectrometry: assessment of matrix effects and assay performance, Scand. J. Clin. Lab. Invest. 70 (2010) 583-591. [47] C. Tron, M. Rayar, A. Petitcollin, J.M. Beaurepaire, V. Cusumano, M.C. Verdier, P. Houssel-Debry, C. Camus, K. Boudjema, E. Bellissant, F. Lemaitre, A high performance liquid chromatography tandem mass spectrometry for the quantification of tacrolimus in human bile in liver transplant recipients, J Chromatogr A. 75 (2016) 55-63.

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[48] D.M. Levine, G.T. Maine, D.A. Armbruster, C. Mussell, C. Buchholz, G. O'Connor, V. Tuck, A. Johnston, D.W. Holt, The need for standardization of tacrolimus assays, Clin. Chem. 57 (2011) 1739-1747.

ACCEPTED MANUSCRIPT List of figures Figure 1. The chemical structures of tacrolimus (TAC; A) and internal standards: TAC13C,D2 (B) and ascomycin (ASC, C).

Figure 2. Comparison of the TAC determinations obtained using TAC13C,D2 or ASC as internal standards

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in pooled (n=18) and spiked (n=35) IPTS samples. Passing-Bablok regression: y = 1.0035 x - 0.028

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presents ASC against TAC13C,D2.

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Figure 3. Comparison of the TAC determinations obtained using TAC13C,D2 or ASC as internal standards

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in pooled (n=18) and spiked (n=35) IPTS samples. Bland-Altman analysis – plot of difference against mean for TAC concentrations (ng/mL) obtained using TAC13C,D2 or ASC expressed as absolute bias.

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Horizontal lines represent bias (solid: mean, dashed: ±1.96 SD).

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Table 1. Within-run and between-run accuracy and precision (n=8) Within-run Concentration declared [ng/mL]

Concentration calculated [ng/mL]*

Accuracy [%]

Between-run Concentration Imprecision calculated [%] [ng/mL]*

Accuracy [%]

Imprecision [%]

Internal standard: ASC 0.5

0.505 ± 0.007

100.99

1.39

0.487 ± 0.018

97.35

3.63

Low QC

1.5

1.52 ± 0.021

101.59

1.39

1.53 ± 0.025

101.71

1.66

Medium QC

7

7.01 ± 0.092

100.08

1.31

99.99

1.65

High QC

16

16.03 ± 0.116

100.16

0.72

100.00

0.77

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LLOQ

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7.00 ± 0.115 16.00 ± 0.123

0.5

0.498 ± 0.005

99.55

1.08

0.501 ± 0.015

100.20

3.09

Low QC

1.5

1.50 ± 0.013

100.26

0.85

1.50 ± 0.039

99.95

2.59

Medium QC

7

7.02 ± 0.055

100.29

0.78

7.02 ± 0.104

100.22

1.48

High QC

16

16.08 ± 0.127

0.79

16.10 ± 0.195

100.63

1.21

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LLOQ

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* mean ± standard deviation.

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Internal standard: TAC13C,D2

100.48

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Table 2. Autosampler stability (n=4) Low QC (~1.5 ng/mL)

High QC (~16 ng/mL) Concentration calculated [ng/mL]*

Stability [%]

Internal standard: ASC 100.00

4

1.53 ± 0.025

100.66

8

1.49 ± 0.028

98.13

12

1.48 ± 0.019

97.29

24

1.48 ± 0.024

97.04

16.17 ± 0.193

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1.52 ± 0.026

16.37 ± 0.291

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initial-0

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Storage time (4ºC) Concentration [h] calculated [ng/mL]*

Stability [%]

100.00 101.24

16.21 ± 0.095

100.22

16.13 ± 0.257

99.72

16.24 ± 0.263

100.40

100.00

16.15 ± 0.183

100.00

4

1.52 ± 0.027

99.35

16.15 ± 0.083

100.03

8

1.49 ± 0.030

97.25

16.19 ± 0.148

100.25

12

1.49 ± 0.036

97.28

16.19 ± 0.108

100.25

24

1.50 ± 0.051

97.98

16.10 ± 0.096

99.69

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1.53 ± 0.024

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initial-0

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Internal standard: TAC13C,D2

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* mean ± standard deviation.

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Table 3. Short-term stability (n=4) Low QC (~1.5 ng/mL) Concentration calculated [ng/mL]*

Concentration calculated [ng/mL]*

Stability [%]

Internal standard: ASC 1.55 ± 0.041

100.00

Stability before preparation procedure

1.53 ± 0.056

99.20

Stability after preparation procedure

1.51 ± 0.088

97.69

15.93 ± 0.209

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Standard analytical procedure

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Procedure description

High QC (~16 ng/mL) Stability [%]

100.00 101.81

15.83 ± 0.319

99.38

100.00

16.21 ± 0.267

100.00

98.50

15.94 ± 0.525

98.29

98.69

15.86 ± 0.388

97.78

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16.22 ± 0.425

1.54 ± 0.057

Stability before preparation procedure

1.51 ± 0.041

Stability after preparation procedure

1.52 ± 0.016

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* mean ± standard deviation.

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Standard analytical procedure

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Internal standard: TAC13C,D2

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Table 4. Long-term stability (n=4) Low QC (~1.5 ng/mL) Storage time (4ºC) Concentration [weeks] calculated [ng/mL]*

High QC (~16 ng/mL) Concentration calculated [ng/mL]*

Stability [%]

Stability [%]

Internal standard: ASC 1.52 ± 0.012

100.00

16.21 ± 0.218

100.00

1

1.52 ± 0.008

100.08

15.99 ± 0.195

98.63

3

1.51 ± 0.010

99.36

4

1.53 ± 0.005

100.92

9

1.53 ± 0.021

100.71

11

1.52 ± 0.022

13

1.51 ± 0.023

15

1.51 ± 0.034

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initial-0

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16.13 ± 0.191

99.46 99.06

16.11 ± 0.238

99.35

100.02

16.00 ± 0.107

98.66

99.62

16.00 ± 0.120

98.67

15.95 ± 0.058

98.35

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16.06 ± 0.205

99.23

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Internal standard: TAC13C,D2

1.51 ± 0.028

100.00

16.07 ± 0.081

100.00

1

1.52 ± 0.022

100.51

15.80 ± 0.220

98.27

3

1.51 ± 0.023

99.92

16.03 ± 0.128

99.72

1.52 ± 0.043

100.83

16.07 ± 0.073

99.97

1.51 ± 0.023

99.92

16.01 ± 0.226

99.60

1.50 ± 0.021

99.06

16.08 ± 0.149

100.03

13

1.49 ± 0.028

98.61

15.98 ± 0.209

99.42

15

1.52 ± 0.033

100.56

16.06 ± 0.295

99.92

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initial-0

* mean ± standard deviation.

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Table 5. Working solution stability (n=3) TAC

TAC13C,D2

ASC

Stability [%]

Concentration calculated [ng/mL]*

Stability [%]

Concentration calculated [ng/mL]*

Stability [%]

initial-0

24.81 ± 0.24

100.00

24.26 ± 0.97

100.00

24.83 ± 0.80

100.00

1

24.34 ± 0.60

98.13

24.19 ± 0.99

99.73

24.36 ± 1.54

98.09

2

25.09 ± 1.24

101.15

24.00 ± 1.82

23.83 ± 0.80

95.98

4

23.76 ± 1.18

95.76

24.17 ± 1.01

24.63 ± 1.17

99.20

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* mean ± standard deviation.

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Storage time (4ºC) Concentration [weeks] calculated [ng/mL]*

99.66

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Table 6. Matrix effects. Low QC (~1.5 ng/mL)

High QC (~16 ng/mL)

Parameter IS

F**

TAC

-32.15 ± 3.92

-31.08 ± 4.34

-1.57 ± 1.74

PE [%] (n=6)*

50.84 ± 2.38

51.85 ± 2.23

97.97 ± 1.57

AR [%] (n=6)*

74.93 ± 3.51

75.24 ± 3.24

-25.98 ± 3.93

F**

-25.74 ± 3.46

-0.37 ± 1.70

55.40 ± 6.97

56.50 ± 7.69

98.16 ± 1.85

99.54 ± 1.60

74.85 ± 9.41

76.08 ± 10.36

98.52 ± 1.86

ME [%] (n=8)*

-16.69 ± 8.76

-18.52 ± 7.92

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ME [%] (n=8)*

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Internal standard: ASC

IS

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TAC

-15.39 ± 6.41

-14.76 ± 7.30

-0.41 ± 1.73

PE [%] (n=6)*

63.48 ± 13.77

62.94 ± 12.22

101.03 ± 11.49 64.74 ± 5.06#

67.76 ± 7.06#

96.01 ± 3.43#

AR [%] (n=6)*

76.20 ± 16.52

77.24 ± 15.00

98.87 ± 11.24

76.52 ± 5.98#

79.49 ± 8.29#

96.40 ± 3.44#

Internal standard: TAC13C,D2

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2.19 ± 1.75

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* mean ± standard deviation; ** F = TAC area / IS area; # n=5 ME –matrix effects; PE – process efficiency; AR – absolute recovery

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Highlights 1. Method parameters are satisfactory for application to pharmacokinetic studies and TDM. 2. Ascomycin presents a performance equivalent to TAC13C,D2 in LC-MS/MS method for TAC.

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3. Matrix effects are successfully compensated by both internal standards.

Figure 1

Figure 2

Figure 3