Analysis of sodium 2-mercaptoethane sulfonate in rat plasma using high performance liquid chromatography tandem-mass spectrometry

Abstract

Sodium 2-mercaptoethane sulfonate (MESNA) is a thiol-containing compound that has proven to be effective in inactivating acrolein, the toxic metabolite of some anti-cancer drugs (e.g., cyclophosphamide and ifosphamide). Also, it scavenges free radicals which cause numerous disorders by attacking biological molecules. Current methods available to analyze MESNA in biological matrices include colorimetry and high-performance liquid chromatography (HPLC) with ultraviolet, fluorescence, or electrochemical detection. These methods have several limitations including low sensitivity, poor selectivity, a high degree of difficulty, and long analysis times. Hence, a rapid, simple, and sensitive HPLC tandem mass spectrometry (MS/MS) method was developed and validated to quantify MESNA in rat plasma following IP administration. The analysis of MESNA was accomplished via plasma protein precipitation, centrifugation, supernatant evaporation, reconstitution, and HPLC-MS/MS analysis. The method showcases an outstanding limit of detection (20 nM), excellent linearity (R²= 0.999, and percent residual accuracy > 90%) and a wide linear range (0.05–200 μM). The method also produced good accuracy and precision (100±10% and <10% relative standard deviation, respectively). The validated method was successfully used to analyze MESNA from treated animals and will allow easier development of MESNA for therapeutic purposes.

1.0. Introduction

The sodium salt of 2-mercaptoethane sulfonate (MESNA) is a thiol-containing drug used to combat urothelial toxicity in patients treated with antineoplastic drugs (e.g., ifosphamide and cyclophosphamide)[1-5] Specifically, some antineoplastic drugs produce toxic side products (e.g., acrolein). MESNA interacts with these metabolites to reduce their toxicity. Because it is important that a chemoprotective agent does not deactivate the potency of anti-cancer drugs, the inability of both MESNA and diMESNA (i.e., oxidized form of MESNA) to passively cross biological lipid membranes due to their hydrophilicity is advantageous since they do not enter cells to disrupt the mechanism of action of anti-cancer drugs.[3] Other uses of MESNA in the medical field include prevention of cisplatin-induced nephrotoxicity by locally reacting with cisplatin species in the kidneys[6-10] and scavenging reactive oxygen species (ROS) due to the ability of its thiol functional group to act as an antioxidant. [2,11-13]

Due to the importance of MESNA in the medical field, several methods have been developed over the years for its bioanalysis, including colorimetry and high-performance liquid chromatography (HPLC) methods using ultraviolet detection (UV),[14] fluorescence detection (FLD),[4] and electrochemical detection (ECD).[15] However, each of these methods has major limitations. Table 1 lists methods for the analysis of MESNA from biological matrices, along with important analytical characteristics. Ellman in 1959 reported the first analysis of MESNA by non-specific colorimetric assay, however the method was not selective enough to distinguish between MESNA and endogenous thiols in biological samples, such as cysteine.[16] To address this limitation, several HPLC techniques were developed to more selectively analyze MESNA from biological samples.[16-19][14]An HPLC-UV technique using 2-chloro-1-methylquinolinium tetrafluoro-borate for precolumn derivatization was reported by Glowacki et al in 2001.[18-20] The method produced excellent accuracy and precision, but suffered from small linear range. Moreover, the selectivity of the method for practical use was limited, with other thiol containing compounds from the biological sample producing similar retention times as MESNA. Verschraagen et al.[21] developed a micro-HPLC method using dual ECD with walled-jacked electrodes to selectively analyze MESNA. Although the method worked to selectively analyze MESNA, the recoveries were poor, the linear range was small, and the analysis time was lengthy. The method also produced poor sensitivity relative to other bioanalytical techniques for analysis of MESNA.[20,22] Mare et al.[2] reported an HPLC-FLD method for analysis of MESNA from plasma and tissues but it requires long sample preparation steps making it inconvenient to analyze large number of samples.[2,23,24]

Table 1.

Comparison of bioanalytical methods for analysis of MESNA.

Reference	Year	Analytical method	Approximate sample preparation and analysis time (min)	R2	Precision (%RSD)	Accuracy (%)	LOD (nM)	Linearity (μM)
Ellman et al[16]	1959	Ellman’s free thiol assay	NR	NR	NR	NR	NR	NR
Glowacki et al. [18]	2001	HPLC-UV	NR	0.997	< 3%	100 ± 3	40	0.16-30
Verschraagen et al.[4]	2003	HPLC-ECD	45	0.999	< 5%	100 ± 6	NR	3–120
Mare et al.[2]	2005	HPLC-FLD	40	0.999	< 10%	100 ± 8.7	1.6	0.0025–2.5
Current technique	2021	HPLC-MS/MS	20	0.999	< 10%	100 ± 10	20	0.05–200

NR = not reported, HPLC = high performance liquid chromatography, UV = ultraviolet (detection), ECD = electrochemical detection, FLD = fluorescence detection, MS/MS = tandem mass spectrometry

Due to the drawbacks of the currently available bioanalytical methods, a highly robust and sensitive method for the analysis of MESNA in biological samples is needed. Therefore, the objective of the current study was to (develop and validate an HPLC-MS/MS method to analyze MESNA from rat plasma.

Materials and Methods

2.1. Materials

All reagents and solvents were at least HPLC grade, unless otherwise stated. Ammonium formate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol and acetone (LC–MS grade) were obtained from Fisher Scientific (Hanover Park, IL, USA). MESNA (>98% purity) and the internal standard, sodium 3-mercapto-1-propane sulfonate (3-MPS, >90% purity), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water used for this study was purified by reverse osmosis and filtered through a Lab Pro polishing unit from Labconco (Kansas City, KS, USA). Stock solutions of MESNA and 3-MPS (10 mM) were prepared in water and stored at 4 °C. The stock solutions of MESNA and 3-MPS were freshly diluted to desired working solutions in water or plasma immediately prior to each experiment.

2.2. Biological Samples

Plasma from Wister Hannover rats with 3.2% sodium citrate anticoagulant (0.2 μm filtered), was purchased from BioIVT for the analytical method development and validation. The plasma was stored at −80 °C until used. To evaluate the ability of the method to detect MESNA in the plasma of treated animals, plasma samples were obtained from MESNA-treated male Sprague-Dawley rats. For this experiment, male Sprague-Dawley rats were obtained from Charles River Laboratories and acclimated at the University of Colorado Denver-Anschutz Medical Campus (AAALAC International accredited) for at least 7 days prior to study. All procedures were approved by the UCD-AMC Institutional Animal Care and Use Committee. A pharmaceutical grade formulation of MESNA (Baxter Healthcare Corporation, NDC 10019-953-01) was administered to rats at 300 mg/kg by intraperitoneal (IP) injection. Control animals received an equivalent volume (3 mL/kg) of saline (Hospira) containing 0.25 mg/mL EDTA (Sigma 03690) by IP injection. At various times following treatment (20 min, 1, 3, or 6 hr), rats were anesthetized by IP injection of a mixture of ketamine (75 mg/kg), xylazine (7.5 mg/kg), and acepromazine (1.5 mg/kg) for blood collection. Blood was collected from the abdominal aorta into 3.2% trisodium citrate anticoagulant (1:9 citrate: blood). Plasma was obtained by centrifugation of blood at 2050 x g for 15 minutes at 4 °C and subsequent transfer of plasma to a clean centrifuge tube (2 mL). The plasma was then flash frozen in liquid nitrogen and shipped on dry ice (overnight) to South Dakota State University. Upon receipt, the plasma was stored at −80 °C until analysis was performed.

2.3. Sample preparation for HPLC-MS/MS analysis

To plasma samples (150 μL), internal standard (100 μL of 10 μM 3-MPS) was added and the mixture was vigorously stirred. Acetone (750 μL) was then added to the plasma to precipitate proteins. The sample was vortexed 3-4 times, and then cold centrifuged (−5 °C) at 13,000 rpm (12,300 × g) for 7 min. An aliquot (900 μL) of the supernatant was transferred into a 4-mL glass screw-top vial and dried at room temperature under nitrogen flow. After drying, the contents of the glass vial were reconstituted with 200 μL of HPLC mobile phase A (10 mM ammonium formate in water). The solution was thoroughly mixed, filtered with a 0.22-μm tetrafluoropolyethylene membrane syringe filter, and transferred into a 2-mL HPLC vial with glass insert (200 μL) for analysis.

2.4. HPLC-MS/MS Analysis

HPLC was performed on a Shimazu UFLC with model LC-20ADXR controller. The column used for chromatography was an Agilent polymeric reversed phase column, Zorbax eclipse-XDB C18 (4.6 × 150 mm, 5.0 μm, part #: PN 993967-902). The chromatographic separation was achieved using isocratic elution at a flow rate of 1 mL/min at 60% B (10 mM ammonium formate in methanol, 5:95) held constant for 5 min. The column was equilibrated for 1 min and 10 μL sample volume was injected for HPLC-MS/MS analysis.

For MS analysis, a tandem mass spectrometer (Sciex Q-Trap 5500 MS) equipped with an electrospray ionization interface in the negative polarity mode was used to detect MESNA. Optimization of MS conditions was accomplished by direct infusion of a standard solution of MESNA into the spectrometer at a flow rate of 10 μL/min. Nitrogen (50 psi) was used as the curtain and nebulization gas. The ion spray voltage was 4,500 V, the source temperature was 500 °C, and both the nebulizer (GS1) and heater (GS2) gas pressures were 90 psi. The collision cell was operated with an entrance potential of 10.0 V and an exit potential of 11.0 V at a “medium” collision gas flow rate. Multiple reaction monitoring mode (MRM) was used for MESNA analysis. Table 2 shows the MS/MS MRM operating parameters for the method. Data analysis was completed using the Analyst software program.

Table 2.

MRM transitions, optimized collision energies (CEs), cell exit potentials (CXPs) and declustering potentials (DPs) for the detection of MESNA and 3-MPS by MS/MS analysis.

Compounds	Q1 (m/z)	Q3 (m/z)	Time (ms)	CE (V)	CXP(V)	DP (V)
MESNA (quantification)	140.40	79.9	100	−25.29	−9.27	−24.24
MESNA (identification)	140.40	138.9	100	−21.20	−9.00	−16.11
3-MPS (quantification)	154.70	80.0	100	−43.25	−14.09	−89.93
3-MPS (identification)	154.70	93.9	100	−24.10	−7.20	−51.02

2.5. Calibration, quantification, and limit of detection

The developed method was validated based on the bioanalytical method development guidelines provided by the Food and Drug Administration (FDA).[3,25,26] The limit of detection (LOD) of MESNA, which is described as the lowest concentration of MESNA that reproducibly produced a signal-to-noise (S/N) of 3, was obtained by analyzing multiple concentrations of MESNA in plasma below the lower limit of quantification (LLOQ) using the developed HPLC-MS/MS method. The noise was calculated as peak-to-peak noise in the blank samples over the retention time of MESNA.

To define the linear range and estimate the accuracy and precision of the method, stock solution of MESNA (10 mM) in water, was diluted in rat plasma to prepare the calibration standards (0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100 and 200 μM) and quality-control (QC) standards (3, 30, and 70 μM). The QC standard concentrations were not included in the calibration range. Each calibration standard was prepared in triplicate and QC standards were prepared in quintuplicate. The calibration curve was plotted using the average peak area signal ratios of MESNA to IS plotted as a function of calibrator concentration. Peak areas of MESNA and IS were obtained by manual integration. To obtain the linearity, accuracy, and precision of the calibration standards, both nonweighted and weighted (1/x and 1/x²) calibration curves were constructed using linear least squares. Since the coefficient of variation (R²) does not accurately quantify goodness-of-fit (GOF) throughout the calibration range,[27] Percent Residual Accuracy (PRA) was used together with the coefficient of determination to determine GOF with PRA values ≥90% indicating an excellent fit.[28,29] A weighted 1/x² fit was chosen as the best model to fit the calibration data based on R² and PRA. The LLOQ and upper limit of quantification (ULOQ) were determined using the inclusion criteria of <15% relative standard deviation (%RSD, as a measure of precision) and a percent error (as a measure of accuracy) of 100±15%. The percent error was calculated based on the comparison of the MESNA concentration back calculated from the calibration curve to the nominal MESNA concentration of the QC standard.

To evaluate the intraassay and interassay accuracy and precision of the method, quality control (QC) standards of 3 μM (low QC), 30 μM (medium QC), and 70 μM (high QC) were analyzed over three days (within 5 calendar days). A calibration curve was constructed each day together with the QCs. Intraassay (within same day) precision and accuracy were calculated for each individual day, and interassay precision and accuracy were calculated by comparing the data gathered over three separate days (over 3 days within 5 calendar days).

2.6. Recovery, matrix effect, and selectivity

Assay recovery of MESNA was determined by analyzing low, medium, and high QC standards of MESNA from spiked plasma and aqueous samples. Low, medium, and high QC standards were prepared in quintuplicate for both matrices. Recovery was determined as the percentage of the average analyte peak area from spiked plasma which went through the sample preparation procedure to the average peak area or signal ratio of aqueous QC standards containing MESNA reconstituted in mobile phase. Internal standard was not used to calculate recovery because the purpose of the recovery experiment was to evaluate incomplete recovery of MESNA during sample preparation. Incorporation of the internal standard in the recovery calculation would correct for loss of MESNA. The matrix effect for MESNA analysis was determined by comparing the slopes (m) of calibration curves created in both aqueous and plasma matrices. The value of the slope ratio (plasma slope/aqueous slope) from the calibration curves was used to quantify the matrix effect. A slope ratio (m_plasma/m_aq) equal to one indicates the matrix does not affect the analysis. On the other hand, m_plasma/m_aq greater than one indicates multiplicative enhancement of the analyte in plasma, whereas slope ratio less than one indicates suppression of the analyte signal by plasma matrix. The efficacy of the IS to correct for the matrix effect was evaluated by comparing the ratio of m_plasma/m_aq of non-IS corrected calibration curves with the IS-corrected calibration curves. To evaluate the selectivity of the method, we confirmed the absence of interfering compounds via evaluation of prominent signals above the baseline in blanks over the retention time of MESNA by comparing several blank rat plasma and MESNA-spiked rat plasma samples.

2.7. Stability

The short- and long-term storage stability of MESNA were evaluated by analyzing low (LQC) and high QC (HQC) MESNA-spiked plasma samples stored at different temperatures and multiple storage times. The stability of MESNA was determined as a ratio of MESNA signal at time(t) to the signal at time “zero”, expressed as a percentage. MESNA was considered stable in plasma at a specific storage condition if the signal was within 10% of the initial signal. For short-term stability of MESNA, both low and high QCs were assessed over 24-hr, both in the autosampler (at 15 and 5°C) and on the benchtop (at room temperature). The autosampler stability was assessed twice on different days by placing triplicates of low and high QCs on the autosampler of the HPLC at 15 and 5°C and analyzing them at approximately 0, 1.5, 2, 4, 8, 12, and 24 hr following preparation. Benchtop stability was evaluated by placing low and high QC plasma samples on the benchtop (room temperature) for 0, 1, 2, 4, 8, 12, and 24 hr and analyzing them after each time point utilizing the procedure above (Section 2.3). The IS was added after storage and prior to sample preparation to correct for the day-to-day instrument signal variation. For freeze-thaw (FT) stability, four sets of LQCs and HQCs were prepared in triplicates without IS. One set of both QCs was analyzed in triplicate immediately, while the other sets were stored at −80 °C. For each cycle, all QC standards were thawed unassisted at room temperature. One set of the thawed QCs was analyzed in triplicate after adding IS to evaluate the first cycle FT stability and the remaining two sets of QCs were again stored at −80 °C. This procedure was repeated twice more to evaluate the remaining two freeze-thaw cycles. A total of three freeze-thaw cycles were evaluated. For the long-term stability studies, QC standards were stored at various storage conditions (−80, −20, 4 °C and room temperature) and analyzed over 30 days (at 0, 1, 5 10, 15, and 30 days). IS was added to each storage stability standard after storage to correct for day-to-day variation in instrument sensitivity.

3. Results and Discussion

3.1. HPLC-MS/MS Analysis of MESNA

The HPLC-MS/MS method developed for analysis of MESNA from rat plasma features quick and simple sample preparation and analysis. The sample preparation is comprised of 4 simple steps: 1) protein precipitation, 2) centrifugation, 3) transfer and drying of the supernatant, and 4) reconstitution in aqueous mobile phase for HPLC-MS/MS analysis. This quick plasma sample preparation is vital to minimize the potential for conversion of MESNA to diMESNA. The simple sample preparation procedure also allows for analysis of about 90 samples within 24-hr period compared to some existing methods. For example, Mare et al.[2] reports an HPLC-FLD method for MESNA utilizing derivatization with Thioglo (TM) 3 solution but requires a 33 min sample preparation procedure and 7 min analysis time. Verschraagen et al.[21] also reports an HPLC-ECD method with sample preparation time of 35 min and chromatographic run time of 12.5 mins.[21] Conversely, our method utilizes a shorter sample preparation and analysis time of 15 and 5 min, respectively. The mass spectra of MESNA and 3-MPS produced by ESI (−)-MS with the most abundant ions identified are shown in Fig. 1A and B, respectively. The m/z ratios of 140.40 and 154.70 correspond to the molecular ions ([M–H]⁻) of MESNA and 3-MPS, respectively. The ([M–2H]) of MESNA and 3-MPS also correspond to 138.9 m/z and 153 m/z respectively. For MESNA, the 140.40→79.9 and 140.40→138.9 transitions were selected as the quantification and identification transitions, respectively. For the internal standard, 3-MPS, the 154.70→80.0 and 154.70→138.9 transitions were selected as the quantification and identification transitions, respectively.

Figure 1.

ESI (−) product ion mass spectra of MESNA (a) and 3-MPS (b) with identification of the abundant ions. Molecular ions of MESNA and 3-MPS [M–H]⁻ correspond to 140.40 and 154.70 m/z, respectively. The ([M–2H]⁻) of MESNA and 3-MPS also correspond to 138.9 m/z and 153 m/z, respectively. Insets, structures of MESNA (a) and 3-MPS (b) with abundant fragments indicated.

HPLC-MS/MS chromatograms of MESNA and its internal standard, 3-MPS, prepared from spiked plasma are shown in Figures 2A and B, respectively. Both peaks eluted at approximately 1.3 min. Our method produced excellent selectivity for both MESNA and 3-MPS with no other prominent peaks present in the chromatograms. The selectivity of the method addresses a disadvantage of multiple methods listed in table.

Figure 2.

Representative HPLC-MS/MS chromatograms of spiked (5 μM) and non-spiked (a) MESNA and (b) 3-MPS (IS) in rat plasma. The chromatograms show signal response to the MRM transitions of MESNA (140.40 → 79.9 and 140.40 → 138.9 m/z) and 3-MPS (154.70 → 80.0 and 154.70 → 93.9 m/z).

3.2. Linear range, calibration, and limit of detection

The linearity of the method was determined by constructing calibration curves over the initial concentration range of 0.05–200 μM. Based on the linear range inclusion criteria for accuracy and precision, the 0.05, 0.1 and 0.2 μM calibrators were excluded. Thus, the linear range of the method was 0.5 μM (LLOQ) to 200 μM (ULOQ) as best defined by 1/x² weighted linear regression. All the calibrators within the linear range had individual PRAs >90% indicating an excellent GOF of the calibration equation for each calibrator. This linear range spanned over three orders of magnitude, which is outstanding for bioanalytical methods.[30,31] This wide linear range is very beneficial when it comes to pharmacological studies where analysis of wide range of MESNA concentrations may be necessary. Additionally, the method showed an excellent LOD of 20 nM.

Calibration curves were constructed over a period of 3 days to determine the consistency of the calibration. As shown in Table 3, the R² and PRAs were consistent and excellent. PRAs were >90% and R² were >0.999 over the period of 3 days but the calibration equation varied significantly. Therefore, the calibration curve should be prepared on the day of analysis.

Table 3.

Linear equations, coefficients of determination (R²), and percent residual accuracy (PRA) for calibration curves created over 3 days.

Day	Equation	R2	PRA (%)
1	y = 10.0428x -1.345	0.9932	96
2	y = 5.8609x - 0.970	0.9943	93
3	y = 9.9436x - 0.511	0.9998	97

3.3. Accuracy and precision

The accuracy and precision of the method were estimated by quintuplicate analysis of low, medium and high QCs on three different days within 5 calendar days (Table 4). The accuracy and precision of the method were excellent for all the 3 days tested. The intraassay and interassay accuracies were both 100±10% of the nominal QC concentrations. The accuracy and precision of the method were remarkable, with all QCs producing %RSDs <10% and accuracies of 100 ± 10% while within the FDA-acceptable range for method validation from a biological matrix.[32-34]

Table 4.

The intra- and interassay accuracies and precisions of MESNA analysis from spiked rat plasma by HPLC-MS/MS.

Conc (μM)	Intraassay						Interassay
	Accuracy (%)			Precision (%RSD)			Accuracy (%)a	Precision (%RSD)a
	Day 1	Day 2	Day 3	Day 1	Day 2	Day 3
3	100±0.4	100±2.0	100±7.0	8.7	4.6	8.8	100±7.0	<8.8
30	100±5.9	100±5.9	100±3.2	9.0	8.6	4.2	100±5.9	<9.0
70	100±1.9	100±9.6	100±0.3	7.8	4.0	6.5	100±9.6	<7.8

^aAggregate of three days of QC method validation (N = 15)

3.4. Matrix effect, recovery, and stability

The evaluation of matrix effects reveals the direct or indirect alteration of response owing to the presence of interfering substances in the plasma matrix. To determine the effect of the plasma matrix on the analysis of MESNA, calibration curves were constructed in both plasma and aqueous samples. The slopes of standard curves of MESNA spiked in aqueous solutions and plasma (m_plasma/m_aq) were compared to obtain the matrix effect value of 0.96, showing only a minor matrix effect for the analysis of MESNA in plasma. The recoveries for low, medium, and high QCs of MESNA in rat plasma were 90%, 91%, and 99% respectively. Addition of internal standard corrected for incomplete recoveries for low, medium, and high QCs to produce 97%, 95%, and 100% signal accuracy, respectively. Since the percent recovery determined in this manner is a combination of matrix effect and recovery, the minor matrix effect in rat plasma does slightly contribute to recoveries less than 100%.

For the benchtop stability, MESNA was stable in rat plasma for the 24-hr period tested. Conversely, MESNA was unstable in prepared samples after 2 hr in the autosampler at 15 °C. Moreover, while samples were stored on the autosampler at 15 °C, both MESNA and the IS signals were inconsistent, leading to the IS not correcting for the loss of MESNA. In an attempt to stabilize the MESNA and 3-MPS in the autosampler, the autosampler was cooled to 5 °C. At this temperature, the stability of MESNA was greatly improved with MESNA producing stable signals for at least 24 hr. Because the IS is added to samples prior to storage on the autosampler, the loss of MESNA signals in the autosampler at 15 °C may be due to the instability of the IS at higher temperatures or reaction of the IS with MESNA at 15 °C. For freeze-thaw stability, MESNA stability was excellent in plasma (−80 °C) over the three freeze-thaw cycles evaluated with not more than 10% deviation from the control. The long-term stability of MESNA was evaluated at multiple storage temperatures. For MESNA samples stored at room temperature and 4 °C, stability was within 10% of the initial signals for 5 days and 2 weeks, respectively, and MESNA signals for both high and low QCs decreased rapidly after 15 days of storage for both storage conditions. Conversely, MESNA showed excellent stability (i.e., not more than 10% deviation from the control) in plasma when the sample was frozen (i.e., at both −20 °C and −80 °C) for at least 30 days (longest time period tested). This is consistent with the results obtained by Verschraagen et al.[35] who showed that MESNA stored at −20°C in deproteinized plasma was stable for at least 35 days. Overall, based on the results of the stability studies, we recommend that when storage is necessary, plasma samples should be stored at either −20 °C or −80 °C and can be thawed and refrozen at least three times for analysis. If samples are to be analyzed within 15 days, they can be stored at 4 °C. Once samples are prepared for the autosampler, they should be kept at 5 °C until analyzed.

3.5. Analysis of MESNA in Treated Animals

The developed method was used to analyze plasma from MESNA-treated rats. Chromatograms from HPLC-MS/MS analysis of the plasma of rats treated with MESNA and rat plasma obtained prior to treatment are shown in Figure 3. In the plasma of treated rats, MESNA was detected as a major peak eluting at 1.30 min. This result shows the applicability of the method for analysis of MESNA-treated animals, and consequently verified the selectivity and usefulness of the method for analysis of MESNA from rat plasma.

Figure 3.

HPLC-MS/MS chromatogram from the plasma of MESNA-treated rats and rat plasma obtained prior to MESNA treatment. The chromatograms represent signal response to MRM quantification transition of (a) MESNA (140.40 → 79.9 m/z) and (b) 3-MPS (154.70 → 80.0).

4.0. Conclusion

A simple and sensitive HPLC-MS/MS method for the determination of MESNA in plasma was developed. The method presented was accurate and precise and showed the ability to analyze MESNA from plasma of treated animals. This technique will allow for simple and rapid analysis of MESNA for further development of this important therapeutic.

Highlights.

• MESNA is a drug used to combat the toxic effects of the anti-cancer agents: cyclophosphamide and ifosfamide.

• A simple and effective method of analysis for MESNA in plasma via HPLC-MS/MS was developed and validated.

• The method has a limit of detection of 20 nM and showcases wide linear range (0.05–200 μM).

• The method was used to detect MESNA from the plasma of MESNA-treated rats.