Simultaneous Quantification of Mesalamine and Its Metabolite N-Acetyl Mesalamine in Human Plasma by LC-MS/MS and Its Application to a Bioequivalence Study

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used for simultaneous quantification of mesalamine and its metabolite N-acetyl mesalamine in human plasma with N-acetyl mesalamine D3 as an internal standard (IS). Chromatographic separation was performed on a Thermo, HyPURITY C18 (150 x 4.6 mm, 5 m) column with an isocratic mobile phase composed of 10 mM ammonium Original Research Article British Journal of Pharmaceutical Research, 4(13): 1568-1590, 2014 1569 acetate and methanol in the ratio of 85:15 (%v/v), at the flowrate of 0.6 mL/min. The drug, metabolite and internal standard were extracted by liquid-liquid extraction. The method was validated over a linear concentration range of 2-1500 ng/mL for mesalamine and 102000 ng/ml for N-acetyl mesalamine, which demonstrated intra and inter-day precision ranging from 1.60 to 8.63% and 2.14 to 8.67% for mesalamine and 0.99 to 5.67% and 1.72 to 4.89% for N-acetyl mesalamine respectively. Similarly, the intraand inter-day accuracy varied from 102.70 to 105.48% and 100.64 to 103.87% for mesalamine, 99.64 to 106.22% and 100.71 to 104.27% for N-acetyl mesalamine respectively. Both analytes were found to be stable throughout freeze–thawing cycles, bench top and postoperative stability studies. The method was successfully applied to support a bioequivalance study of healthy subjects.


INTRODUCTION
Mesalazine (INN, BAN), also known as mesalamine (USAN) or 5-aminosalicylic acid (5-ASA), is an anti-inflammatory drug used to treat inflammation of the digestive tract (Crohn's disease) and mild to moderate ulcerative colitis. Mesalazine is a bowel-specific aminosalicylate drug that is metabolized in the gut and has its predominant actions there, thereby having fewer systemic side effects. As a derivative of salicylic acid, 5-ASA is also an antioxidant that traps free radicals, which are potentially damaging by-products of metabolism. The major metabolite of mesalamine (5-aminosalicylic acid) is N-acetyl-5aminosalicylic acid or N-acetyl mesalamine. Its formation is brought about by Nacetyltransferase activity in the liver and intestinal mucosa [1,2]. The recommended dosage for the induction of remission in adult patients with active, mild to moderate ulcerative colitis is two to four 1.2g tablets to be taken once daily with meal for a total daily dose of 2.4g or 4.8g [3,4]. Treatment duration in controlled clinical trials was up to 8 weeks. The total absorption of mesalamine from Lialda® 2.4g or 4.8g given once daily for 14 days to healthy volunteers was found to be approximately 22% of the administered dose. Mesalamine is approximately 43% bound to plasma proteins at the concentration of 2.5 µg/mL. Mesalamine is mainly eliminated of via the renal route following metabolism to N-acetyl-5-aminosalicylic acid (acetylation). However, there is also limited parent drug excreted in urine. Of the approximately 22% of the dose absorbed, less than 8% of the dose compared with greater than 13% for N-acetyl-5-aminosalicylic acid was excreted unchanged in the urine [5][6][7].
The main purpose of the present study is to develop and validate simple extraction method (LLE), high sensitive (5 times higher than that proposed by Gu GZ et al. [12], rugged and reproducible bioanalytical method. At the same time suitable deuterated internal standard was used to compare analytes. Finally, the method was used to compute the pharmacokinetic parameters of two brands of mesalamine 400mg enteric coated tablets and then to evaluate the bioequivalence between the two Mesacol ® (SUN Pharma Ltd., India) was selected as the reference produce, while APL Research Centre brand (Pv.T., Ltd, India) as the test formulation.

Instrumentation and Conditions
The LC system was manufactured by (Agilent Technologies, model series 1200, Waldbronn, Germany). Mass spectrometric detection was performed on an API 4200 triple quadrupole instrument (ABI-SCIEX, Toronto, Canada). Data were processed on Analyst 1.5.1 software package (SCIEX). Turbo ion spray (IS) negative mode with Unit Resolution, MRM was used for the detection. For mesalamine and N-acetyl mesalamine the [M-H] were monitored at m/z 152.0 and m/z 194.2 as the precursor ion, and a fragment at m/z 108.0 and m/z 149.9 as the product ion, respectively. For internal standard N-Acetyl mesalamine D3 the [M-H] was monitored at m/z 169.9 as the precursor ion and a fragment at m/z: 153.0, as the product ion. Mass parameters were optimized as source temperature 650ºC, nebulizer gas 20 psi, heater gas 30 psi , curtain gas 30 psi, CAD gas 4 psi, IS voltage -2000 volts, source flow rate 600 µL/min without split, entrance potential 10 V, Collision cell exit potential (CXP) 12 V, Declustering potential (DP) 50 V, collision energy (CE) 35 V for mesalamine, N-acetyl mesalamine, and N-Acetyl mesalamine D3, respectively.

Chromatographic Condition
Chromatography was performed on a Thermo, Hypurity-C18 (150 x 4.6 mm, 5 m) column. The mobile phase used as 10mM ammonium acetate: methanol, 85:15 v/v at the flow rate of 0.6 mL/min. Injection volume was 10µL, column temperature was 40ºC with an isocratic elution mode.

Sample Preparation
Liquid-liquid extraction was used for extraction of drug and IS. For this purpose, an 100 μL internal standard solution (150 ng/mL of N-Acetyl mesalamine-D3) was added to 100 µL of plasma sample (respective concentration) into riavial. To this, 25 µL of derivatisation solution(10% propionic anhydride in methanol) was added and vortexed briefly. After that, 100 µL of 0.5% formic acid was added into each tube and vortexed briefly again. Then, 3 mL of methyl t-butyl ether was added and vortexed for 10 minutes. Samples were then centrifuged for 5 minutes at 4000 rpm at 20ºC. Supernatant from each sample was transferred into ria vial and evaporated to dryness. This was followed by reconstitution with 800 µL of reconstitution solution (10 mM ammonium acetate : methanol, 85:15 v/v). and vortex briefly. From this, 5 μL of sample was injected into the LC-MS/MS system through the autosampler.

Selectivity and specificity
The selectivity of the method was determined by six different human blank plasma samples, which were pretreated and analyzed to test the potential interferences of endogenous compounds co-eluting with analyte and IS. Chromatographic peaks of analyte and IS were identified based on their retention times and MRM responses. The peak area of mesalamine and N-Acetyl mesalamine at the respective retention time in blank samples should not be more than 20% of the mean peak area of LLOQ of mesalamine and N-Acetyl mesalamine. Similarly, the peak area of N-Acetyl mesalamine -D3 at the respective retention time in blank samples should not be more than 5% of the mean peak area of LLOQ of N-Acetyl mesalamine-D3.

Matrix effect
To predict the variability of matrix effects in samples from individual subjects, matrix effect was quantified by matrix factor, which was calculated as Peak response ratio in presence of extracted matrix (post extracted) to peak response ratio in aqueous standardsSix lots of blank biological matrices were extracted each in triplicates and post spiked with the aqueous standard at the Mid QC level, and compared with aqueous standards of same concentration. The overall precision of the matrix factor is expressed as coefficient of variation (CV in %) should be < 15%.

Linearity, precission and accuracy
The calibration curve was constructed using values ranging from 2.0 to 1500.0 ng/mL for mesalamine and 10.0 to 2000.0 ng/mL for N-Acetyl mesalamine in human plasma respectively. Calibration curve was obtained by linear model with weighted 1/x 2 regression analysis. The peak area ratio of mesalamine / N-Acetyl mesalamine-D3 was plotted against the mesalamine concentration in ng/mL for mesalamine. The peak area ratio of N-Acetyl mesalamine / N-Acetyl mesalamine-D3 was plotted against the N-Acetyl mesalamine concentration in ng/mL for N-Acetyl mesalamine. Calibration curve standard samples and quality control samples were prepared in replicates (n=6) for analysis. Precision and Accuracy for the back calculated concentrations of the calibration points, should be within ≤15 and ± 15% of their nominal values. However, for LLOQ, the Precision and Accuracy should be within ≤ 20 and ± 20%.

Recovery
The extraction recovery of mesalamine / N-Acetyl mesalamine and N-Acetyl mesalamine -D3 from human plasma was determined by analyzing quality control samples. Recovery at three concentrations (6, 450 and 1050 ng/mL for mesalamine and 30,600 and 1400 ng/mL for N-Acetyl mesalamine) was determined by comparing peak areas obtained from the plasma sample and those from the standard solution spiked with the blank plasma residue. A recovery of more than 50% was considered adequate to obtain required sensitivity.

LOQ and LOD
The response (peak area) was determined in blank plasma samples (six replicates from different plasma) and spiked LOQ sample prepared from the same plasma was determined. The peak area of blank samples should not be more than 20% of the mean peak area of LOQ of mesalamine/ N-Acetyl mesalamine and not more than 5% of N-Acetyl mesalamineD3. The precision and mean accuracy of the back calculated LOQ replicate concentrations must be ≤ 20 and ± 20%, respectively. The limit of detection (LOD) is a parameter providing the lowest concentration in a sample that can be detected from background noise but can not be quantitated. LOD was determined using the signal-to-noise ratio (s/n) of 3:1 by comparing test results from samples with known concentrations of analyte with blank samples.

Stability
Low quality control and high quality control samples (n=6) were retrieved from deep freezer after three freeze-thaw cycles according to the clinical protocols. Samples were stored at −30ºC in three cycles of 24, 36 and 48 h. In addition, the long-term stability of mesalamine / N-Acetyl mesalamine in quality control samples was also evaluated by analysis after 62 days of storage at −30ºC. Autosampler stability was studied following 48 h storage period in the autosampler tray with control concentrations. Bench top stability was studied for period of 25 h with control concentrations. Stability samples were processed and extracted along with the freshly spiked calibration curve standards. The precision and accuracy for the stability samples must be within ≤ 15 and ± 15% of their nominal concentrations, respectively. Stability of the mesalamine / N-Acetyl mesalamine, N-Acetyl mesalamine-D3 in stock solution was evaluated for 9 days with comparision of freshly prepared stock solutions. Similarly stability of N-Acetyl mesalamine-D3 working solution was also proved for 9 days with comparision of freshly prepared working solutions

Study subjects
The study was carried out in accordance with the current revision of the Declaration of Helsinki concerning medical research in humans. Study protocol was approved by IEC (Institutional Ethics committee) as per DCGI (Drug control general of india). Thirty four healthy male subjects were included in the study. All volunteers gave a written informed consent prior to participation, after they had been thoroughly informed and they understood the nature and details of the study. All clinical laboratory tests were performed by the ISO 15189 certified laboratories. The daily results of the clinical laboratory tests including the quality control data were verified by its own independent quality assurance personnel before reporting. Subject inclusion criteria included Indian male, aged between 18-45 years, no consumption of drugs or food supplements for 4 weeks prior to the study, and no participation in any bioavailability or bioequivalence study at least 30 days prior to the present study.

Study design
The study was conducted as an open label, randomized two-period, two-sequence, singledose crossover bioequivalence study under fasting condition, and a wash-out period of 14 days. All subjects arrived at the clinical research laboratory, at least 12 h prior to the start of the study. They were housed in an air-conditioned facility and were given a standard dinner, which was finished at least 10 h before dosing in each period of the study. On the day of drug dosing in period 1, volunteers were randomly assigned to one of two treatment sequences TR (sequence 1) or RT (sequence 2), as indicated in a pre-printed randomization scheme, Subjects in sequence 1 received treatment T at the first dosing period and then crossed over to receive treatment R at the second dosing period (after the 7-day washout period). Subjects in sequence 2 received treatments in the order of R and T at the two dosing periods. The subjects administered the assigned mesalamine formulation with 240 mL of plain drinking water. This was followed by an oral cavity check to ensure completion of the dose administration. Subjects were required to refrain from lying down during the first 4 h after intaking the tablet. No meal was permitted until 4 h after dosing. Drinking water was restricted from 1 h before dosing till 2 h after excess water intake (> 100 mL/h) was not permitted. Lunch, snacks, and dinner were served as per the scheduled time. All subjects abstained from any xanthine-containing food or beverages for at least 72 h and alcoholic products for at least 7 days prior to formulation administration and throughout the sampling schedule during each period. No concomitant medication was permitted during the study period.

Pharmacokinetics and statistical analysis
Pharmacokinetic parameters from the human plasma samples were calculated by a noncompartmental statistic model using Win Nonlin 5.0. software (Pharsight, USA). Blood samples were taken for a period of 3 to 5 times the terminal elimination half-life (t 1/2 ) and it was considered as the area under the concentration time curve (AUC) ratio higher than 80% as per USFDA guidelines. Plasma mesalamine, N-Acetyl mesalamine concentration-time profiles were visually inspected, and C max and T max values were determined. The AUC 0-t was obtained by the trapezoidal method. AUC 0-∞ was calculated up to the last measureable concentration and extrapolations were obtained using the last measureable concentration and the terminal elimination rate constant (K e ) was estimated from the slope of the terminal exponential phase of the plasma of the mesalamine, N-Acetyl mesalamine concentrationtime curve (by linear regression method [35,36,37]. The terminal elimination half-life (t 1/2 ), was then calculated as 0.693/K e [38]. Regarding AUC 0-t, AUC 0-∞ and C max bioequivalence were assessed by means of analysis of variance (ANOVA) and calculating the standard 90% confidence intervals (90% CIs) of the ratio's test/reference (logarithmically transformed data). The bioequivalence was considered when the ratio of averages of log transformed data was within 80-125% for AUC 0-t, AUC 0-∞ and C max .

Method Development
LC-MS/MS has been recognized as one of the most powerful analytical techniques in clinical pharmacokinetics for its selectivity, sensitivity and reproducibility [12]. The goal of this work was to develop and validate a simple, sensitive and rapid assay method for the quantitative determination of mesalamine, N-acetyl mesalamine from plasma samples. A simple liquidliquid extraction technique was utilized in the extraction of mesalamine, N-acetyl mesalamine and N-Acetyl mesalamine-D3 from the plasma samples. Chromatographic conditions, especially the composition and nature of the mobile phase, were optimized through several trials to achieve best resolution and increase the signal of mesalamine, N-acetyl mesalamine and N-Acetyl mesalamine-D3. The MS optimization was performed by direct infusion of solutions of both mesalamine, N-acetyl mesalamine and N-Acetyl mesalamine D3 into the ESI source of the mass spectrometer.
Other parameters, such as the nebulizer and the heater gases were optimized to obtain a better spray shape, resulting in better ionization. mesalamine, N-acetyl mesalamine and N-Acetyl mesalamine-D3 were detected with proton adducts at m/z 152.0108.0, 194.2149.9 and 169.9153.0 in multiple reaction monitoring (MRM) negative mode respectively (Fig. 2a-2b, Fig. 2c-2d, and Fig. 3a-3b). After the MRM channels were tuned, the mobile phase was changed from more organic phase to an aqueous phase to obtain a fast and selective LC method. A good separation and elution were achieved using the proposed LC study condition.

Selectivity and specificity
The analysis of mesalamine, N-acetyl mesalamine and N-Acetyl mesalamine-D3 using MRM function was highly selective with no interfering compounds. Fig. 4 shows the chromatograms of one blank human plasma. Chromatograms obtained from plasma spiked with mesalamine (2.0 ng/mL), N-Acetyl mesalamine (10.0 ng/mL) and N-Acetyl mesalamine D3 (150 ng/mL) are shown in (Figs. 4 and 5).

Matrix effect
Six lots of blank biological matrices were extracted each in triplicates and post spiked with the aqueous standard at the mid QC level, and compared with aqueous standards of same concentration in alternate injections. The overall precision of the matrix factor is 2.83 for mesalamine, and 2.80 for N-Acetyl mesalamine, respectively. There was no ionsuppression and ion-enhancement effect observed due to IS and analyte at respective retention time.

Linearity, precision and accuracy
Calibration curves were plotted as the peak area ratio (Mesalamine / N-Acetyl mesalamine-D3) versus (mesalamine) concentration for Mesalamine and the peak area ratio (N-Acetyl mesalamine / N-Acetyl mesalamine-D3) versus (N-acetyl mesalamine) concentration for Nacetyl mesalamine. Calibration was found to be linear over the concentration range of 2-15000 ng/mL for mesalamine and 10-2000 ng/mL for N-Acetyl mesalamine. The Precision (CV %) for mesalamine, N-Acetyl mesalamine was less than 8.67% and 5.67% respectively. The accuracy ranged from 100.64 to 105.48% for mesalamine and 98.55 to 106.22% for N-acetyl mesalamine. The determination coefficients (r 2 ) for mesalamine and N-acetyl mesalamine were greater than 0.9998 and 0.9987 respectively for all curves ( Table 2).
Precision and accuracy for this method was controlled by calculating the intra and interbatch variations of QC samples in six replicates at four concentrations (2, 6, 450 and 1050 ng/mL) for mesalamine and (10, 30, 600 and 1400 ng/mL) N-Acetyl mesalamine respectively as shown in (Table 3).

Fig. 5. LOQ Chromatograms of mesalamine and N-Acetyl mesalamine
This method was demonstrated intra and inter-day precision 1.60 to 8.63 and 2.14 to 8.67% for mesalamine 0.99 to 5.67 and 1.72 to 4.89% for N-Acetyl mesalamine. This method demonstrated intra and inter-day accuracy 102.70 to 105.48 and 100.64 to 103.87% for mesalamine, 99.64 to 106.22 and 100.71 to 104.27% for N-acetyl mesalamine. These results indicate the adequate reliability and reproducibility of this method within the analytical range.

Recovery
The recovery following the sample preparation using Liquid-Liquid extraction method with tbutyl methyl ether was calculated by comparing the peak areas of drug in plasma samples with the peak area ratios of solvent samples and was estimated at control levels of drug. The recovery of mesalamine (at concentrations 6,450 and 1050 ng/mL) and N-acetyl mesalamine (at concentrations 30,600 and 1400 ng/mL) were found to be 95.97, 91.79, 98.87% and 88.27, 89.24,90.16%, respectively. The overall mean recovery of mesalamine, N-Acetyl mesalamine, and N-Acetyl mesalamine-D3 were 95.54%, 89.22% and 86.71% respectively.

LOQ and LOD
The limit of quantification for this method was proven as the lowest concentration of the calibration curve which was proven as 2.00 ng/mL for mesalamine and 10.00 ng/mL for Nacetyl mesalamine. The LOD was determined using aqueous solutions. For mesalamine 10 L of a 1.0 pg/mL solution was injected to give an on-column mass of 10.0 Femtogram (fg) and for N-acetyl mesalamine, 10 L of a 2.0 pg/mL solution was injected to give an oncolumn mass of 20.0 fg respectively.

Stability
Quantification of the mesalamine, N-acetyl mesalamine in plasma subjected to 3 freezethaw (from -30ºC to room temperature) cycles showed the stability of the analyte and its metabolite. The accuracy ranged from 99.33 to 100.64% for mesalamine and 98.10 to 102.11% for N-Acetyl mesalamine of the theoretical values. No significant degradation of mesalamine and N-Acetyl mesalamine was observed even after 48 h storage period in the autosampler tray and accuracy of mesalamine and N-Acetyl mesalamine were between 98.50 and 96.86% and 100.95 and 105.72% of the theoretical values. The room temperature stability of mesalamine and N-Acetyl mesalamine in QC samples after 25 h was also evaluated and accuracy ranged from 98.25 to 98.61% for mesalamine and 99.17 to 102.67% for N-Acetyl mesalamine of the theoretical values. In addition, the long-term stability of mesalamine, N-acetyl mesalamine in QC samples after 62 days of storage at −30ºC was also evaluated and accuracy ranged from 97.94 to 102.54% for mesalamine and 102.26 to 106.73% for N-Acetyl mesalamine of the theoretical values. These results confirmed the stability of mesalamine and N-Acetyl mesalamine in human plasma for at least 62 days at -30ºC ( Table 4). Stability of the mesalamine and N-acetyl mesalamine in stock solution was proved for 9 days, and N-Acetyl mesalamine-D3 working solution was proved for 9 days with freshly prepared stock solutions and working solutions.

Application to Biological Samples
The above validated method was used in the determination of mesalamine and N-Acetyl mesalamine in plasma samples for establishing the bioequivalence of a single 400 mg dose (one 400 mg tablet) in Thirty four healthy volunteers. Typical plasma concentration versus time profiles is shown in (Figs. 6 and 7). All the plasma concentrations of mesalamine and N-Acetyl mesalamine were within the standard curve region and retained above the 2.0 ng/mL as the LOQ of mesalamine and above 10.0 ng/mL as the LOQ of N-Acetyl mesalamine for the entire sampling period (Tables 5 and 6). The ANOVA results revealed that period, sequence and treatment had no statistically significant effects on C max , AUC 0-tlast and AUC 0-∞ . Since the sequence or carry-over effect was not significant, the ANOVA test was valid. The statistically significant subject within sequence effect on C max , AUC 0-tlast and AUC 0-∞ were observed that are usually seen in large sample size study as in crossed over bioequivalence studies. Bioequivalence between the 400 mg enteric coated tablet formulations of mesalamine under fasting condition was demonstrated by the 90% CI of the geometric mean ratios of C max , AUC 0-tlast and AUC 0-∞ lying within the acceptable criteria of 80-125%. The test and reference formulations had very similar t 1/2 at approximately 9.5 h for mesalamine and 15.5h for N-Acetyl mesalamine. Period, sequence and treatment had no significant effects on C max , AUC 0-tlast and AUC 0-∞ .

CONCLUSION
A method has been developed and validated over the concentration range of 2 -1500 ng/mL for mesalamine and 10 -2000 ng/mL for N-Acetyl mesalamine in human plasma. The selectivity, sensitivity, precision and accuracy obtained with this method enabled to test the present study. The validated method was successfully applied in 34 healthy volunteers and demonstrated the bioequivalence of the 400 mg mesalamine enteric-coated tablet formulations of test product (APLRC) as well as the reference product (Mesacol ® ) .The results concluded that the two formulations can be used interchangeably.

CONSENT
Not applicable.

ETHICAL APPROVAL
All authors hereby declare that all experiments have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.