A robust procedure for determination of immunosuppressants Cyclosporine A and Tacrolimus in blood samples with detection of LC-MS/MS

doi:10.21203/rs.3.rs-1986533/v1

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Research Article

A robust procedure for determination of immunosuppressants Cyclosporine A and Tacrolimus in blood samples with detection of LC-MS/MS

https://doi.org/10.21203/rs.3.rs-1986533/v1

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published 19 Feb, 2023

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Therapeutic monitoring of immunosuppressants is a key tool in the treatment of transplant patients. It is essential to develop accurate methods to obtain precise and reliable results. a simple protein precipitation method with absence of zinc sulfate was explored to pretreat blood samples, the influence of matrix was eliminated, Cyclosporine A and Tacrolimus were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The linearity, matrix effect, carry over, accuracy, intraday and interday precision of this method were investigated. The linear relationship of tacrolimus and cyclosporine were 1–40 ng/mL and 50-2000 ng/mL, respectively; The correlation coefficient (R²) was both greater than 0.995. The intraday and interday precision of cyclosporine and tacrolimus were ≤ 10%. The lower limit of quantitation (LLOQ) of tacrolimus and cyclosporine A were 1 ng mL^− 1 and 50 ng mL^− 1, respectively. The main features of this method are simplicity, promptness, robustness, high sensitivity and throughput and good selectivity. It is suitable for therapeutic drug monitoring of cyclosporine A and tacrolimus in routine clinical diagnostics.

liquid chromatography tandem mass spectrometry (LC-MS/MS)

immunosuppressant

cyclosporine A

tacrolimus

In the field of solid organ transplantation, cyclosporine A and tacrolimus are most commonly used immunosuppressive agents, because they have great potential to prevent allograft rejection and decrease patient morbidity and mortality.^1–3 However, due to their narrow therapeutic window and high drug toxicity, excessive immunosuppressant can induce a series of side effects, while insufficient dosage of immunosuppressant or low drug concentration in blood may lead to organ rejection.^{1, 4–6} The adverse effects of cyclosporine are mainly nephrotoxicity and hypertension.^{2, 7} For recipients with increased blood pressure after postoperative application of cyclosporine or hypertension, available treatments include taking oral antihypertensive drugs, combining with mycophenolate mofetil to reduce the dose of cyclosporine, or switching to tacrolimus. In addition, adverse effects caused by cyclosporine also include hepatotoxicity, neurotoxicity, hypercholesterolemia, hyperuricemia, hyperkalemia, tremor, gingival hyperplasia, diabetes and hypertrichosis. The main side effects of tacrolimus are nephrotoxicity, neurotoxicity and diabetes. Other side effects of tacrolimus include tremor, bacterial infection, cytomegalovirus infection and gastrointestinal reaction.

Particularly, immunosuppressive agents show a great degree of inter-individual and intra-individual pharmacokinetic and pharmacodynamic variability.^{5, 8, 9} Moreover, the age, weight, combined medication, food and fluid intake of a patient can alter the metabolization of the drugs.^{10, 11} Therefore, it is of great significance for transplant recipients to do therapeutic drug monitoring (TDM) regularly to maintain the concentration of immunosuppressant within the therapeutic range and minimize the toxic effects and risk of acute rejection after organ transplanted.¹²

Up to now, there are many available analytical methods in therapeutic drug monitoring, e.g., microparticle enzyme immunoassay (MEIA), enzyme multiplied immunoassay technique (EMIT), fluorescent polarization immunoassay (FPIA), high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS).^{5, 13}

Immunoassay results tend to show significant positive bias due to cross-reactivity with metabolites, similar-structure drug or heterophilic antibodies.^{4, 10, 14, 15} LC-MS/MS is considered as the gold standard method for monitoring the above mentioned immunosuppressants^{10, 16, 17} because of its high specificity and sensitivity.¹⁸ In addition, LC-MS/MS is able to simultaneously measure several drugs in one single analytical run.¹⁹

However, many LC-MS/MS methods adopt the pretreatment methods of liquid-liquid extraction and solid-phase extraction, which is time-consuming and complex.³ And other LC-MS/MS methods commonly used zinc sulfate (ZnSO₄) and methanol or acetonitrile as protein precipitant.^20–22 Zinc sulfate is a nonvolatile inorganic salt which is difficult to remove after entering the mass spectrum. The instrument is easy to get dirty, which increases the frequency of maintenance and cleaning. Here we exhibit a rapid and reliable LC-MS/MS method, which does not use zinc sulfate and has broad linearity range covering the full therapeutic ranges of the analyzed drugs. The pretreatment procedures are extremely simple and do not involve time- and money-consuming steps.

Chemicals and stock solutions

Tacrolimus, cyclosporin A were purchased from Sigma-Aldrich (USA). The internal standards (IS) tacrolimus-13C and cyclosporin-d12 were purchased from Cmass (China). HPLC grade acetonitrile and methanol were obtained from Merck (Germany). LC-MS grade formic acid (FA) was purchased from Fisher scientific (USA). LC-MS grade ammonium formate was purchased from Fisher Chemical (Germany). A millipore purification system was used to produce ultrapure water (Merck Millipore).

Calibration standards and quality control samples

Starting from a 1 mg mL^− 1 concentration for tacrolimus, first, 50 µg mL^− 1 sub-stock solution of tacrolimus was prepared by further diluting the original stock solution using acetonitrile. Then 40 µL sub-stock solution of tacrolimus and 100 µL cyclosporine A original stock solution was added to 860 µL of 50% methanol to prepare the working calibration standards which containing 2000 ng mL^− 1 of tacrolimus (TAC) and 100000 ng mL^− 1 of cyclosporine A (CsA). This solution was further diluted by 50% methanol to 7-point calibrators: 20.0, 40.0, 80.0, 160.0, 320.0, 600.0, 800.0 ng mL^− 1 for TAC and 0.0, 2000, 4000, 8000, 16000, 30000, 40000 ng mL^− 1 for CsA. For the purpose of method validation only, QC working solutions of TAC and CsA were prepared in the same way but from different stock solutions. Three concentration levels prepared were 60.0, 180.0, 640.0 ng mL^− 1 for TAC and 3000, 9000, 32000 ng mL^− 1 for CsA, respectively.

On the day prior to analysis, 5 µL aliquoted working solutions for calibrators and QCs were thawed to spike to 95 µL drug-free human whole blood. 7-point calibrators(S1-S7)acquired were 1, 2, 4, 8, 16, 30, 40 ng mL^− 1 for tacrolimus and 50, 100, 200, 400, 800, 1500, 2000 ng mL^− 1 for CsA, respectively. LQC, MQC, HQC acquired were 3, 9, 32 ng mL^− 1 for tacrolimus and 150, 450, 1600 ng mL^− 1 for CsA, respectively.

70 µL cyclosporine A-d12(concentration 100 µg mL) and 30 µL tacrolimus-13C (concentration 100 µg mL^− 1) were placed into a 100 mL volumetric flask and flushed with LC–MS acetonitrile. A mixture of 30 ng mL^− 1 tacrolimus-13C and 70 ng mL^− 1 cyclosporine A-d12 were used as the working internal standard solution.

Commercially available quality controls (ClinChek® Whole Blood Controls, Germany) are used for internal quality assurance in routine clinical examination. Concentrations are 3.49, 7.11, 14.4 ng mL^− 1 for tacrolimus and 51.0, 105, 204 ng mL^− 1 for cyclosporine A, respectively. Quality controls were implemented before and after sample batch every day.

All stock solutions and working solutions were stored at -70°C and light-protected until analysis. Batches were stable for at least 6 months. One batch of calibrators and quality control samples was thawed 10 min before sample preparation.

Sample preparation

For the analysis of EDTA-anticoagulated patient blood samples, whole blood samples were shaken absolutely. Consequently, 100 µL of the samples were transferred into 1.5 mL Eppendorf tubes. Followed by the addition of 400 µL acetonitrile containing internal standard for CsA and TAC to precipitate protein. Then, they were vortexed for 10 s, sonicated for 10 s and centrifuged for 10 min at 10000 rpm. Sonication was critical to guarantee adequate recovery of the immunosuppressants by breaking down sample components. Finally, 200 µL supernatant was transferred to 600 µL water in a sample bottle. After vortexing for 4 s, it was placed in the autosampler and an aliquot of 6 µL was injected into the LC–MS system.

Instrumentation and LC-MS/MS method

A 4500MD triple quadrupole mass spectrometer (AB SCIEX, for in vitro Diagnostic use) coupled with Jasper LC System and peak gas generation (Genius AE 32) was used to determine the concentration of immunosuppressants in the whole blood. Data processing was performed on Analyst MD 1.6.2 Software.

Chromatographic separation was achieved using a Kinetex® 2.6 µm C18 100Å 50×2.1 mm column (Phenomenex, Torrance, USA). The column oven was maintained at a temperature of 60°C. The autosampler temperature was kept at 15°C.

Mobile phase A consisted of 2 mM ammonium acetate with 0.1% formic acid (v/v) in water, which was also used as a weak wash solvent. Mobile phase B consisted of 2 mM ammonium acetate with 0.1% formic acid (v/v) in methanol, which was also used as a strong wash solvent. 50% Methanol (v/v) was used as a seal wash. The mobile phase flow rate was maintained at 0.3 mL min^− 1. From zero to 0.4 min, isocratic conditions were run with 55% B. Solvent B was increased from 55 to 98% in the time range from 0.4 to 0.9 min. Then, solvent B was kept at 98% for 2 min. The mobile phase reverted to 55% B from 2.9 min to 2.95 min. Re-equilibration was performed from 2.95 to 4.5 min at 55% B. The total instrumental analysis time was 4.5 min.

Mass spectrometric conditions

The mass spectrometer was operated in positive electrospray source ionization mode (ESI+). Detection parameters applied to both whole blood samples were as follows: curtain gas 30.0 µL/min, collision gas 7 mL/min, the source temperature was set at 400°C, the ion spray voltage at 5500 V, ion source gas 1: 50 psi and ion source gas 2: 50 psi. Entrance Potential (EP)10.0, Collision Cell Exit Potential (CXP) 7.0. For each immunosuppressant, two transitions were followed: one of them was used for quantification (the quantifier), and the other was monitored for identification (the qualifier). The quantifier to qualifier ratio was used for peak identification based on criteria set forth by CLSI C62-A guidelines.

Multiple reaction monitoring (MRM) transitions for each compound are summarized in Table 1.

Table 1

MRM transitions and compound-specific MS parameters
Compound	Parent ion(m/z)	Daughter(m/z) ion(m/z)		DP voltage(v)	CE voltage(ev)
TAC	821.700	768.500	45.0	95.000	31.000
TAC	821.700	786.700	45.0	95.000	25.000
CsA	1220.000	1203.000	45.0	130.000	46.000
CsA	1203.000	1185.000	45.0	130.000	56.000
CsA-IS	1215.100	1084.000	45.0	110.000	59.000
TAC-IS	826.700	612.500	45.0	130.000	50.000

Analytical method validation

The method validation of whole blood was based on the European Medicines Agency (EMA), U.S. Food and Drug Administration (FDA) guidelines on bioanalytical method validation and C62-A. EDTA-anticoagulated blood samples from healthy volunteers were used as blank whole blood. The following performance parameters were evaluated: linearity, selectivity, carryover, lower limit of quantitation, recovery, matrix effect, the intraday and interday precision, accuracy and stability. In addition, the experiment was validated using external quality assessment (EQA) program, and passed with a score of 100. Results: All performance parameters were within acceptance criteria, and hence, it was determined that the validated method is fit for clinical routine analysis.

Linearity

To establish the relationship between the concentration of the analytes and the detector response, 7-point calibrators were prepared for two immunosuppressants, covering the entire range of expected concentrations in patient blood samples.

Linearity was evaluated by linear regression. Each calibrator was injected on three consecutive days. Response was defined as the peak area ratio of the analytes to the internal standards (y) and was plotted against the nominal concentration (x) of each drug. The weighting of 1/x² was chosen for both analytes^, calibration curves, the trueness and precision of the lower limit of quantitation (LLOQ) were < 15% bias and CV < 20%, the trueness and imprecision of other calibrators should not exceed 15%.

Table 2 collect the values for linear regression results, linear range and R² of CsA and TAC in human blood, respectively.

Table 2

Linearity results of CsA and TAC
Analyte	Concentration (ngmL^− 1)	Day 1		Day 2		Day 3
Analyte	Concentration (ngmL^− 1)	RE%	R²	RE%	R²	RE%	R²
TAC	1.00	-0.77	0.9975	-4.49	0.9977	0.41	0.9994
	2.00	-3.37		6.00		-0.85
	4.00	10.51		5.95		-0.62
	8.00	-0.26		4.65		-0.56
	16.0	2.27		-6.16		5.78
	30.0	-8.48		-5.28		-1.44
	40.0	0.1		-0.66		-2.71
CsA	50	3.11	0.9985	1.85	0.9955	3.00	0.9982
	100	-6.96		-6.43		-7.76
	200	-1.87		5.82		4.79
	400	6.42		1.82		-1.89
	800	2.15		-4.02		-3.08
	1500	-3.47		-10.89		-1.06
	2000	0.62		11.84		6.01

Specificity/selectivity and carry-over

Specificity/selectivity means the capability of differentiating or selecting the targeted analyte from its complex matrix. As shown in Fig. 1, in daily clinical routine calibrating, Double Blank (no analyte, no IS) and Blank(no analyte, with IS)samples were injected before injecting seven calibrators. According to the EMA guideline, the absence of interfering components is accepted when the peak area response of interfering peak at the retention time of analyte (each immunosuppressant in our case) is less than 20% of the LOQ for the analyte and 5% for the IS.

Table 3

Analytes and IS peak area of three days
		Day 1		Day 2		Day 3
		Analyte Peak Area	IS Peak Area	Analyte Peak Area	IS Peak Area	Analyte Peak Area	IS Peak Area
CYC A	DB	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00
	BL	0.00E + 00	5.30E + 04	0.00E + 00	4.30E + 04	0.00E + 00	4.63E + 04
	S1	5.26E + 03	5.74E + 04	4.01E + 03	4.45E + 04	4.78E + 03	4.63E + 04
	S7	2.25E + 05	5.31E + 04	1.78E + 05	3.98E + 04	1.86E + 05	4.53E + 04
	DB	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00
TACR	DB	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00
	BL	0.00E + 00	2.95E + 04	0.00E + 00	2.67E + 04	0.00E + 00	2.70E + 04
	S1	3.64E + 03	2.86E + 04	3.42E + 03	2.62E + 04	3.37E + 03	2.69E + 04
	S7	1.38E + 05	2.96E + 04	1.33E + 05	2.64E + 04	1.34E + 05	2.64E + 04
	DB	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00	0.00E + 00

In order to assess carryover, a Double Blank (no analyte, no IS) sample was injected immediately after the highest calibrator. Criteria for acceptability included that the blank sample area following the highest calibrator should not be > 20.0% of the LLOQ area for each immunosuppressant and > 5.0% for the internal standards.

Lower limit of quantification (LLOQ)

The LLOQ defined as the lowest point of a calibration curve was the lowest concentration of analyte that gave precision and accuracy values within the limits of condition (CV ≤ 20%, bias < 15%), and a signal-to-noise ratio (S/N) of at least 10.²³ It was then determined experimentally by analyzing six replicates samples in three batches during different days. Calculated the average value, bias and CV. According to concentration of the immunosuppressants in the literature, CsA and TAC were clinically found at 150–1000 ng mL^− 1 and 4–70 ng mL^− 1 in human blood of transplanted patients, respectively,^{24, 25} the LLOQ of this method can meet the clinical demand.

Table 4

Detection of LLOQ
Compound	Theoretical concentration	Sample	Average	Bias (%)	Precision (CV, %)	S/N
TAC	1 ng mL^− 1	18	1.056	5.60%	8.7	196.5
CsA	50 ng mL^− 1	18	52.017	4.03%	8	260.6

Extraction recovery

To evaluate the loss of sensitivity caused by sample pretreatment. According to the sample preparing procedure, three levels of QC samples in blank whole blood matrix were processed, n = 3 for each group, and the peak area A was acquired. Then, the blank whole blood without adding internal standards were processed in accordance with the sample preparing method. Consequently, a certain amount of IS and QC (LQC\MQC\HQC) was spiked into the supernatant, n = 3 for each group. Subsequently, the peak area B was acquired by injecting the samples into the instrument. The recovery can be defined as the ratio of the peak area response of adding standard before matrix extraction (Area A) to the peak area response of adding standard after matrix extraction (Area B) ¹².

Also, the internal standard extraction recovery was calculated the same as the analytes.

Table 5

The extraction recoveries determined of the immunosuppressants
Compound	QC	Average value of Area A	Average value of Area B	Recovery (%)	CV (%)
TAC	LQC	1.06E + 04	1.38E + 04	76.81	2.50
	MQC	3.20E + 04	4.25E + 04	75.29
	HQC	1.22E + 05	1.52E + 05	80.26
CsA	LQC	1.83E + 04	1.91E + 04	95.8	3.20
	MQC	5.34E + 04	5.94E + 04	89.9
	HQC	1.99E + 05	2.14E + 05	93.1
Recovery after sample extraction resulted in a coefficient of variation (cv) of 2.5% for TAC, 3.2% for CsA.

Matrix effect

The matrix components of whole blood generally include salts, lipids, proteins, organic small molecules and so on. Salts and lipids (especially phospholipids) are the most important factors affecting the ionization efficiency. Most matrix interferences can be removed by sample pretreatment or chromatographic separation. Matrix effect was assessed by analyzing blank whole blood samples from six different healthy volunteers as six different matrix sources. First, according to the sample preparing method, samples were pretreated without adding internal standards, and then a certain amount of IS and (LQC/HQC) was spiked to the supernatant. And the peak area B was acquired by injecting the samples into the instrument. Determine same amount of (LQC/HQC) and IS in the pure solution, and the peak area C was acquired, n = 3 for each group, calculate the average value.

Quantitative measurement of the matrix effect can be defined as the ratio of the peak area response in the presence of the matrix (Area B) to the peak area response in pure solution of analyte (Area C). Calculate the matrix factor and the cv% of 6 matrixes.

Also, the IS-normalized matrix effect was calculated as same as the analytes.

Table 6

Matrix effects of TAC and CsA
	TAC			CsA
	Average value of Analyte Area	Area Ratio	Matrix effect (cv%)	Average value of Analyte Area	Area Ratio	Matrix effect (cv%)
ME-L-1	1.25E + 04	94.0%		1.61E + 04	95.1%
ME-L-2	1.42E + 04	106.5%		1.71E + 04	101.2%
ME-L-3	1.26E + 04	95.0%	6.00%	1.55E + 04	91.7%	7.10%
ME-L-4	1.37E + 04	103.0%		1.67E + 04	99.0%
ME-L-5	1.27E + 04	95.7%		1.49E + 04	88.0%
ME-L-6	1.42E + 04	107.0%		1.81E + 04	107.1%
Solution-L	1.33E + 04			1.69E + 04
ME-H-1	1.49E + 05	101.1%		1.84E + 05	100.9%
ME-H-2	1.47E + 05	99.8%		1.84E + 05	101.3%
ME-H-3	1.49E + 05	101.1%	2.1%	1.92E + 05	105.3%	3.7%
ME-H-4	1.48E + 05	100.2%		1.88E + 05	103.5%
ME-H-5	1.44E + 05	98.0%		1.72E + 05	94.3%
ME-H-6	1.54E + 05	104.3%		1.85E + 05	101.5%
Solution-H	1.47E + 05			1.82E + 05

According to the CLSI C62-A, the variability in matrix effect as measured by the coefficient of variation (cv) should be less than 15%, so this method can solve matrix problems.

The within-day and between-day precision

The within-day precision was measured by replicate analysis (n = 6) of the three levels of QC samples in whole blood matrix during each analytical run. The between-day precision was determined by evaluating the QC samples during three different analytical runs on three consecutive days. The acceptance criterion for the bias was 15%, except for the LLOQ, where it was 20%.

Table 7

The within-day precision and between-day precision
Compound	Within day (%)	Between day (%)	Compound	Within day (%)	Between day (%)
TAC(LLOQ)	6.7	8.7	CsA(LLOQ)	4.7	8.0
	9.7			9.3
	7.9			7.4
TAC(QC1)	3.8	5.6	CsA(QC1)	7.9	6.5
	7.7			5.2
	4.4			7.1
TAC(QC2)	3.0	5.4	CsA(QC2)	6.2	5.9
	5.5			3.8
	4.3			7.9
TAC(QC3)	5.5	5.5	CsA(QC3)	7.8	8.6
	6.9			7.9
	4.6			8.5

For the two immunosuppressive drugs, the within-day and between-day precision were both within the acceptable limits stated for bioanalytical method validation. According to these results, the assay is accurate and precise enough for the studied concentration range.

Stability

The stability of the samples included the stability of the unprocessed whole blood samples and extracted samples in the autosampler. According to the actual situation of the laboratory, it demands a short intra-laboratory turn-around time. Patient samples were detected as soon as possible, except fault diagnosis and maintenance when samples were analyzed within 48 h. So, samples stability within 72 h were assessed. Unprocessed whole blood samples were stored in refrigerator (2–8℃) for a period of 3 days with an absolute percent deviation from the nominal concentration lower than 10% for CsA, 12% for TAC. Compared the concentration of extracted samples in the autosampler with the nominal concentration, an absolute percent deviation from the nominal concentration lower than 11% for CsA, 10% for TAC for a period of 3 days.

Proficiency Testing

To compare the performance against other laboratories using the similar method, the laboratory has participated in the external quality assessment (EQA) program (organized by National Center for Clinical Laboratory).

Table 8

Proficiency testing results of CsA
Number	Measurement value	Target value	Bias (%)	Permitted range	Performance
202211	378.7	410.1	-7.6%	307.6- 512.6	pass
202212	290.5	266.0	9.21	199.5-332.5	pass
202213	112.7	112.6	0.09	84.5-140.8	pass
202214	368.6	340.4	8.28	255.3-425.5	pass
202215	218.5	207.3	5.40	155.5-259.1	pass

Table 9

Proficiency testing results of TAC
Number	Measurement value	Target value	Bias (%)	Permitted range	Performance
202211	12.3	13.1	-6.11	9.8–16.4	pass
202212	7.8	8.0	-2.50	6.0–10.0	pass
202213	2.9	2.9	0.00	2.2–3.6	pass
202214	10.3	10.7	-3.74	8.0-13.4	pass
202215	6.3	6.2	1.61	4.7–7.8	pass

In this work, we have develop a fast, simple and robust LC-MS/MS method for the determination of CsA and TAC. The sample pretreatment procedures are extremely simple, low cost and high throughput, which would be practical for implementation in a clinical laboratory for the routine TDM. Until now, more than 20000 patient samples were detected in our clinical laboratory. The high selectivity of LC-MS/MS methods prevents an overestimation of the concentration of immunosuppressive agents in patient samples. Therefore, LC-MS/MS techniques can provide precise service for clinical diagnostics.

Acknowledgment

The authors appreciate the financial support of Project Clinical Immunosuppressant program. The authors want to acknowledge the technical and human support provided by adicon clinical laboratory and Department of Chemistry, Northeastern University.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was supported by Clinical mass spectrometry R&D.

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25 Aug, 2022
First submitted to journal
22 Aug, 2022

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A robust procedure for determination of immunosuppressants Cyclosporine A and Tacrolimus in blood samples with detection of LC-MS/MS

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Chemicals and stock solutions

Calibration standards and quality control samples

Sample preparation

Instrumentation and LC-MS/MS method

Mass spectrometric conditions

Results And Conclusion

Analytical method validation

Linearity

Specificity/selectivity and carry-over

Lower limit of quantification (LLOQ)

Extraction recovery

Matrix effect

The within-day and between-day precision

Stability

Proficiency Testing

Conclusion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1