INTRODUCTION:

Tamsulosin hydrochloride (TAM) is (-) - (R)- 5 - [ 2- [ 2 - (o - Ethoxy phenoxy) ethyl] amino ] propyl ] - 2 - methoxy benzene sulfonamide monohydrochloride (Figure 1) ¹.

TAM is a selective α₁-adrenoreceptor antagonist prescribed for the treatment of patients with symptomatic benign prostatic hyperplasia (BPH) ². The rapid absorption of the immediate-release oral TAM formulations has led to the development of modified-release once daily formulation to improve tolerability and prolong the drug action. TAM is available in single or in combination with 5α-reductase inhibitors such as finasteride and dutasteride³.

Since oral TAM formulations contain a low dose of the drug (0.4 mg TAM), the manufacturing process is important to ensure optimum efficacy of the dosage form. Reports in clinical practice have shown the lack of therapeutic effectiveness of some marketed TAM formulations due to inappropriate manufacturing processes ⁴. Several analytical methods for the determination of TAM in dosage forms have been reported. These include spectrometric⁵, voltametric⁶, HPLC^7-8 and HPTLC⁹. Recently, electrospray liquid chromatography-tandem mass spectrometry [LC-MS/MS] has emerged as a powerful tool for the quantitative determination of TAM in biological fluids¹⁰. Although the reported HPLC procedures were accurate and precise, however, the majority of the described methods was tedious, time consuming and needed a complete chromatographic separation of TAM and IS. Recently, LC-MS/MS has been recognized as a potential technique for the analysis of drugs, metabolites and biomolecules^11-12 due to specificity, sensitivity and high sample throughput. This study discusses the in vitro evaluation of some selected brands of TAM formulations using a developed LC-MS/MS method. The study reports on the determination of TAM contents in capsules or tablets in six different brands and highlights the evaluation of the in vitro dissolution behaviors of the examined brands under simulated gastric conditions.

Tamsulosin (TAM) Procainamide (IS)

Fig. 1: Chemical structures of tamsulosin and procainamide (internal standard, IS)

MATERIALS AND METHODS:

MaterialsReference powdersof tamsulosin hydrochloride (purity ≥ 98%, Sigma, USA) and procainamide hydrochloride (Sigma, USA) (internal standard) were used (Figure 1). Tablets and capsules of different TAM brands were purchased from local and overseas sources. All solvents were of HPLC grade and the reagents were of analytical grade. Milli-Q purified water (Millipore, Bedford, MA, USA) was used throughout this work.

LC-MS/MS instrument and conditionsA triple quadruple tandem mass spectrometer (Quattro LC, Micromass, UK) fitted with a Z-spray ion source and positive electrospray (ES) ionization probe was used. The system was coupled to Waters 2695 Separation Module, Alliance HPLC and Waters autosampler. The chromatographic analyses were conducted using MS XTerra^® RP-C₈ (150 x 4.6 mm, 5 μm) column and a mobile phase of acetonitrile: water: formic acid (80:20:50μl v/v/v). Prior to use, the mobile phase was filtered through a 0.2 µm Millipore membrane filter and degassed by sonication. The flow rate of the mobile phase was 0.4 ml/min. The volume of injection was 20 μl and the run time was 6 minutes. The tuning parameters for analysis of TAM and IS by +ESI-MS/MS were optimized using Harvard Infusion pump.The MS tuning parameters for capillary voltage, cone voltage and collision energy were selected as 3.5 kV, 25 V, and 30 eV, respectively for optimum detection of TAM and IS. The source and desolvation temperatures were adjusted to 100^oC and 250^oC, respectively.

Method validation:

Linearity:Stock solutions of TAM and IS were separately prepared at concentrations (1mg/ml) in methanol and stored at 4^oC. TAM calibrators were prepared using TAM working solution (0.25 µg/µl) to obtain the concentrations 1, 5, 10, 15, 20 and 25 µg/ml, respectively. The solutions were mixed with 20 µl of IS working solution (0.01 µg/µl) to obtain a concentration of 20 µg/ml. The MRM transitions at m/z 410.4>148.6 (TAM) and m/z 236.8>120.4 (internal standard) were selected for quantification of TAM by the internal standard calibration method. System operation and data acquisition were controlled by MassLynx software. The linear regression equations representing the calibration curves were automatically calculated using MassLynx quantifying program. The linearity was confirmed by calculating the means of slopes and correlation coefficients of at least 20 calibration curves over the concentrations 1-25 µg/ml.

Precision:The intra-day precision (expressed as RSD%) was assessed by replicate analysis of quality control TAM solutions at 5, 10, 20 µg/ml concentrations in the same day (n=10). Inter-day precision was established by the analysis of one sample from each QC solution (n=10) on ten different days.

Accuracy:The intra-day and inter-day accuracy (expressed as percentage deviation from theoretical concentrations, bias %) were evaluated for TAM solutions at 5, 10, 20 µg/ml concentrations as shown above.

Limits of quantification (LOQ) and detection (LOD):LOQ was experimentally determined on the basis of the lowest concentration of TAM on the calibration curve that produces RSD% < 25%.The LOQ and LOD could be also calculated from the linearity curve using the formulas:

LOQ= 10X[Residual standard deviation/Slope]

LOD= 3X[Residual standard deviation/Slope]

Recovery studies:Recovery percentages of TAM were determined for the quality control (QC) samples containing TAM at 15 µg/ml concentration (n=20). Recoveries of TAM were computed by comparing the found and theoretical concentrations.

Content uniformity:Individual capsules or tablets of each TAM brand were sonicated with 25 ml of the mobile phase for 120 minutes. The mixture was filtered using Millipore membrane filter (0.45 µm) and approximately 0.98 ml aliquot of the clear filtrate was mixed with 20 µl of IS solution and a 20 µl volume was injected into the LC-MS/MS. The concentration of TAM was determined by the MassLynx quantifying program using weighting 1/x least squares linear regression analysis. The TAM content in each dosage form was determined as the weight in milligrams of TAM per capsule or tablet. Student t-test was used to assess any significant differences among the examined brands in terms of content uniformity (n=6).

Dissolution study:Dissolution procedures of individual capsules or tablets of different TAM brands were conducted in 0.1M HCl solution at 37 ± 2^oC and 120 rpm (simulated gastric fluid). Aliquots from the dissolution media were withdrawn at 0.0, 15.0, 30.0, 45.0, 60.0, 90.0, 105.0 and 120.0 minutes, filtered using membrane filter (Millipore, 0.45 µm) and approximately 0.98 ml aliquots of the clear filtrates were mixed with 20 µl aliquots of IS solution and 20 µl volumes were injected. Each drawn sample volume was replaced by fresh aliquots of 0.1M HCl solution to maintain the volume of the dissolution medium. The concentrations of TAM in solutions were determined by the instrument quantifying program in reference to the calibration curves and were used to establish the dissolution plots. The average % drug release of TAM ± SD from capsules or tablets were calculated for each brand (n=6). Student t-test was used to assess any significant differences among the examined brands in terms of % drug release of TAM from dosage form.

RESULTS AND DISCUSSION:

Development of LC-MS/MS method:

Under the optimized mass spectrometer tuning parameters, TAMand IS were detected at the molecular mass ions m/z410.4 and 236.8, respectively using a positive electrospray ionization(+ESI) (Figures 2 and 3). In daughter (MS/MS) mode, TAM and IS produced the daughter mass ions at m/z 148.6 and 120.4, respectively ( Figures 2 and 3). The mass ions pairs were used to establish the MRM transitions at m/z 410.4>148.6 (TAM) and 236.8>120.4 (IS) for quantitative analysis using internal standard calibration (Figure 4).

Fig. 2: Full-scan positive electrospray mass spectrum (A) and daughter mass ion spectrum (B) of TAM

Fig. 3: Full-scan positive electrospray mass spectrum (A) and the daughter mass ion spectrum (B) of procainamide (IS)

Fig. 4: MRM ion-chromatograms of a mixture containing TAM (5 µg/ml) and IS (20 µg/ml)

The composition of the mobile phase was optimized to achieve good detection and resolution of the analytes at a short run-time. A mobile phase of acetonitrile/water/formic acid in a ratio 80/20/0.05 v/v/v was appropriate. The presence of formic acid has improved the ionization and detection of TAM and IS by +ESI. The high percentage of organic modifier permits rapid elution of analytes. A reversed-phase C₈ column improved the peak shape and allowed elimination of the ion suppression effect. Under the selected chromatographic conditions of the mobile phase and column, TAM and IS were rapidly eluted at retention time ≤ 4 min, as well-defined peaks. As the detection of TAM and IS was based on monitoring of their specific MRM transitions, therefore a complete chromatographic separation of TAM and IS was not required. The elimination of the ion suppression effect was indicated by the absence of peak interference at the retention times of TAM and IS in MRM chromatograms of a drug-free sample (Figure 5).

Fig. 5: MRM ion-chromatograms of a drug- and IS-free sample

Validation of the Method:

The developed LC-MS/MS method was validated in accordance to international guidelines¹³. The method was statistically validated for linearity, limit of quantification, intra-day and inter-day precision and accuracy and recovery %. The linearity was established by using internal standard calibration. As the utilization of the isotope-labeled TAM as internal standard was not feasible, several compounds of similar chemical properties were tested. Procainamide hydrochloride was found to be appropriate (Figure 1) as the chemical and ionization properties of the internal standard are similar to TAM hydrochloride. Calibration curves constructed from the peak area ratio (TAM/IS) and TAM concentrations were linear over the concentration range 1-25 µg/ml using linear regression analysis. The mean values of slope and intercept ± SD were 9.1 ± 1.1 and 9.9 ± 2.4, respectively (Table 1). The correlation coefficient was 0.995 ± 0.003 (RSD%: 0.30) and the LOQ and LOD were found to be 1.0 µg/ml and 0.39 µg/ml, respectively pointing towards adequate sensitivity of the method. The intra-day and inter-day precision (RSD% ˂ 10.8%), accuracy (Bias% ˂ +1.4%) and recovery % (99.2 ± 5.47) were within the acceptable limits (Table 2). The obtained validation data supported the application of the developed method for content uniformity and dissolution studies.

Table No. 1:Linearity of the developed LC-MS/MS method for the analysis of TAM

Linearity(n=20) Parameter	Mean ± SD
Correlation coefficient:	0.995 ± 0.003	(RSD%*: 0.30)
Intercept:	9.9 ± 2.4	(RSD%: 24.2)
Slope:	9.1 ± 1.1	(RSD%: 12.1)
Limit of Quantification (LOQ) (n=20)	1.00 ± 0.241 µg/ml	(RSD%: 24.1)
Limit of Detection (LOD) (n=20)	0.39µg/ml

^*RSD% : Relative Standard Deviation

Table No.2: Precision, accuracy and recovery data for the determination of TAM by the developed LC-MS/MS method

Intraday Precision (n=10) Concentration of TAM (µg/ml)	Mean ± SD	RSD%
5	5.1 ± 0.54	10.65
10	10.1 ± 0.50	4.95
20	20.1 ± 0.60	2.98
Intraday Precision (n=10) Concentration of TAM (µg/ml)	Mean ± SD	RSD%
5	5.03 ±0.54	10.80
10	10.01± 0.44	4.40
20	20.07± 0.77	3.84
Intraday Accuracy (n=10) Concentration of TAM(µg/ml)	Bias%*	% Found
5	+1.40	101.4
10	+1.00	101.0
20	+0.50	100.5
Intraday Accuracy (n=10) Concentration of TAM(µg/ml)	Bias%*	% Found
5	+0.60	100.6
10	+0.10	100.1
20	+0.35	100.4
Recovery study (n=20) Concentration of TAM(µg/ml)	Recovery % ± SD
15 µg/ml	99.2% ± 5.47

*Bias%= [Found concentration -Theoretical concentration] / Theoretical concentration

Content uniformity study:

Six capsules or tablets of each brand were used for the content uniformity study.The analysis indicated that TAM contents of brands I-VI formulations were in the range of 0.295-0.379 mg. Statistical analysis of the data using student t-test indicated that the differences of TAM contents between Brand V (reference brand) and the generic brands II and III were insignificant; whereas significant differences between brand V and the generic brands I, IV and VI were detected (Table 3). Figure 6 showed the MRM ion chromatogram of the extracted TAM compound from brand V capsules.

Table No. 3: TAM contents in brands I-VI capsules or tablets (0.4 mg TAM) as determined by LC-MS/MS method (n=6).

Brand	Mean ± SD	Calculated t-value*
Brand I	0.349 ± 0.045	5.19^b
Brand II	0.365 ± 0.018	0.23
Brand III	0.351 ± 0.044	0.11
Brand IV	0.295 ± 0.019	14.55^b
Brand V^a	0.379 ± 0.015	-----
Brand VI	0.328 ± 0.013	8.97^b

Brand I: Contiflo^® OD(0.4 mg Tamsulosin Hydrochloride Extended Release Capsules, Ranbaxy, India); Brand II: Tamsulin^® (0.4 mg Tamsulosin Hydrochloride Capsules, Marcyrl Pharmaceutical Industries, Egypt); Brand III: Tamurex^® (0.4 mg Tamsulosin Hydrochloride Prolonged Release Capsules, Somex Pharma, United Kingdom); Brand IV: Omnic^® OCAS (0.4 mg Tamsulosin Hydrochloride Oral Controlled Association System (OCAS) Film Coated Tablet, SAJA Pharmaceuticals, KSA); Brand V: Bazetham^® MR (0.4 mg Tamsulosin Hydrochloride Modified Release Capsules, PLIVA Pharma, United Kingdom); Brand VI: Omnic^® (0.4 mg Tamsulosin Hydrochloride Modified Release Capsules, SAJA Pharmaceuticals, KSA)

^aReference Brand

^b Significant

*Theoretical t-value is 2.23 at P=0.05

Dissolution study:

Six capsules or tablets of each brand (reference and generic) were used for the dissolution study using LC-MS/MS to determine the concentration of TAM. The analysis indicated variable dissolution behaviors among TAM brands under simulated gastric conditions (0.1M HCl) (Figure 7). The % drug release of TAM for controlled-release formulations (brands I, III, V, VI) was in the range of 40.49-53.83% within 2 hours, whereas for immediate-release formulation (Brand II), the % drug release was found to be 89.19 % (Table 4). As indicated, the obtained results for the controlled-release formulations were in good agreement with the reported values¹⁴. The reference brand V exhibited the highest dissolution profile (53.83 ± 5.58), whereas the generic brand IV exhibited a very low % drug release (4.16 ± 3.02) under the same experimental conditions. Statistical analysis of data using student-t-test indicated insignificant differencesbetween brand V and brands III and VI, whereas significant differences between brand V and I or IV were found (Table 4). The significant low % drug release of TAM from brand IV capsules in addition to low drug content might be attributed to formulation problems.

Fig. 7: Dissolution profiles of six brand formulations in simulated gastric fluid (0.1M HCl) at 37± 2^oC(n=6)

Table No. 4: % Drug release of TAM from brands I-VI formulations in 0.1M HCl solution at 37± 2^oC (n=6)

Brand	% Drug Release ± SD
Brand I	40.49 ±11.66 (t=2.48)*^b
Brand II	89.19 ± 6.06
Brand III	46.81 ± 5.58 (t=0.87)*
Brand IV	4.16 ± 3.02 (t=18.7)*^b
Brand V^a	53.83 ± 5.58
Brand VI	41.42 ± 14.18 (t=1.08)*

Brand I : Contiflo^® OD(0.4 mg Tamsulosin Hydrochloride Extended Release Capsules, Ranbaxy, India); Brand II: Tamsulin^® (0.4 mg Tamsulosin Hydrochloride Capsules, Marcyrl Pharmaceutical Industries, Egypt); Brand III: Tamurex^® (0.4 mg Tamsulosin Hydrochloride Prolonged Release Capsules, Somex Pharma, United Kingdom); Brand IV: Omnic^® OCAS (0.4 mg Tamsulosin Hydrochloride Oral Controlled Association System (OCAS) Film Coated Tablet, SAJA Pharmaceuticals, KSA); Brand V: Bazetham^® MR (0.4 mg Tamsulosin Hydrochloride Modified Release Capsules, PLIVA Pharma, United Kingdom); Brand VI: Omnic^® (0.4 mg Tamsulosin Hydrochloride Modified Release Capsules, SAJA Pharmaceuticals, KSA).

* Values in parenthesis are the calculated t-values (theoretical t-value: 2.23) at P=0.05

^a Reference brand

^bSignificant

CONCLUSION:

The developed +ESI LC-MS/MS method proved to be successful for monitoring TAM at low doses (0.4 mg) for content uniformity and dissolution studies. The appropriate sensitivity and selectivity of the method, in addition to high throughput capability permitted a rapid and accurate determination of TAM in different brands. The obtained results proved that the content uniformity assessments as well as dissolution studies are important for pre-formulation studies and for judgment of the in vitro bioavailability of different brands of TAM before conducting in vivo bioavailability study. Furthermore, these studies might reflect the poor therapeutic efficacy of some generic brands of TAM in comparison to the reference branded product.

ACKNOWLEDGEMENT:

The authors are grateful to the Faculty of Pharmacy, Kuwait University for providing the research facility of LC-MS/MS instrument used in this work.

REFERENCES:

1. The Merck Index, Merck Research Laboratories, Division of Merck andCo., Inc., Whitehouse Station, NJ, 2006; 1728.

2. Sweetman Sean C(ed) Martindale; The Complete Drug Reference, Pharmaceutical Press, The Royal Pharmaceutical Society of Great Britain, 2005.

3. Woo H. The effects of dutasteride or tamsulosin alone and in combination on storage and voiding symptoms in men with lower urinary tract symptoms (LUTS) and benign prostatic hyperplasia (BPH): 4-year data from the combination of avodart and tamsulosin (CombAT) study.BJU Int.2011;107(9):1431. doi: 10.1111/j.1464-410X.2011.10128.x

4. www.dancewithshadows.com/pillscribe/us-fda-stops-ranbaxys-generic-tamsulosin-flomax-due-to-poor-compliance-of-gmp-in-manufacturing-plants

5. Shrivastava A, Saxena P and Gupta VB. Spectrometric estimation of tamsulosin hydrochloride by acid-dye method. Pharmaceutical Methods 2011;2(1): 53-60. doi: 10.4103/2229-4708.81089

6. Ozkan SA, Uslu B and Aboul-Enein HY. Voltammetric investigation of tamsulosin. Talanta 2003; 61 (2): 147-156.

7. Aboul-Enein HY,Hussein RF,Radwan MA,Yusuf A,Al-Ahmadi W andAl-Rawithi S.Tamsulosin dissolution from pharmaceutical dosage forms using an automated HPLC system.J Liquid Chrom and Related Tech 2003; 26 (7): 1109-1116.

8. Wang YH, Cheng ZZ, Gao Y and Li H.HPLC determination of content and related substances of tamsulosin hydrochloride and its sustained release capsules. Yaowu Fenxi Zazhi 2008; 28(12): 2055-2058.

9. Choudhari VP and Nikalje APG.Stability-indicating HPTLC method for the determination of tamsulosin in pharmaceutical dosage forms. Chromatographia 2009; 69(11-12): 1463-1467.

10. Ding L, Li LM, Tao P, Yang J and Zhang ZX.Quantitation of tamsulosin in human plasma by liquid chromatography-electrospray ionization mass spectrometry.J Chromatography B: Anal. Technol. Biomed. Life Sci. 2002; 767(1): 75-81.

11. Niessen WMA. Liquid Chromatography-Mass Spectrometry. 2nd ed., Marcel Dekker, INC, New York, USA, 1998.

12. Voyksner RD. Electrospray Ionization Mass Spectrometry; Fundamentals, Instrumentation and Applications: Cole R.B (ed). Wiley Interscience, New York, 1997.

13. Guidance for Industry, Bioanalytical Method Validation.U.S. Department of Health and Human Services Food and Drug Administration.Federal Register. 2001; 66.

14. www.freepatentsonline.com/EP1784161.pdf

Received on 21.09.2011 Modified on 11.10.2011

Research J. Pharm. and Tech. 4(12): Dec. 2011; Page 1885-1890