Development, Validation and Application on Seized materials by UV Spectrophotometric Method for the determination of a Solid Semi-legal Drug: Tramadol Hydrochloride Case

 

Salifou Karimoune Fadjimata, Adamou Rabani, Adamou Hassane Hassane, Ayouba Mahamane Abdoulkadri

Materials, Water and Environment Laboratory (MWEL), Department of Chemistry, Abdou Moumouni University. BP 10662, Niamey (Niger).

*Corresponding Author E-mail: salifoufadjimata@gmail.com

The present study describes development and validation of simple, fast, non-toxic and economic UV spectrophotometric method for the determination of Tramadol Hydrochloride in bulk, capsules and tablet dosage form using absorbance maxima method. Solubility studies indicated that a Tramadol Hydrochloride shows good solubility in proposed diluent solution as compared to solubility in hydrochloride acid and the λmax of Tramadol Hydrochloride was found to be 271 nm. Distilled water, was selected like diluent with λmax at 271 nm and the high peak time was found to be 20 min. The developed method was validated for linearity, precision, accuracy. The LOD and LOQ were calculated as per protocol for the validation of a method of analysis in chemistry guidelines, they are respectively be 0.177 µg mL^-1 and 0.592 µg mL^-1. This proposed method was found to be linear with R² = 0.9996 in the concentration range of 1-120 µg/ml. It is reproducible in this range and can be used in case of possible Tramadol seizures or quality controls. In all cases like intraday and Interday, the % RSD were less than 2. The ratio of similarly was found to be 5.71. The standard was found to be 100%. The present method was found to be simple, linear, precise, accurate and sensitive, and can be used for in laboratories for the quantitative analysis. It also can be preferred because of cost-effectiveness, non-toxicity and could be compared to other UV spectrophotometric validated methods.

 

 

 

1.    INTRODUCTION

Tramadol hydrochloride is one of the most used drug in Africa usually and specifically in Niger for injured person. More than 60 tons has been seized in 2016¹. It is a major analgesic classified in the category of semi-legal drugs and the family of opiates. It comes in several forms (liquid, capsules and tablets) whose most seized forms are forms of tablets and capsules because it is easy to conceal them in the sale of counterfeit medicines². Its chemical formula is C₁₆H₂₆ClNO₂. It has two enantiomeric forms, which according to UIPAC are: (1R, 2R) -rel-2- (dimethylamino) methyl-; 1- (3-methoxyphenyl) cyclohexanol and (1S, 2S) -rel-2- (dimethylamino) methyl-; 1- (3-methoxyphenyl) cyclohexanol³. A review of the literature reveals that, up to now, several methods of tramadol analysis have been validated and proposed such as HPLC⁴, UV spectrophotometr0y alone or combination of other methods have been reported for the estimation of tramadol hydrochloride^5,6,7,8,9. These methods are used only for the determination of tramadol hydrochloride or it may be in combination with other drugs. Among these methods, however, only a few included a single estimate of tramadol hydrochloride^{3,10, 11, 12}. The main objective of the proposed methods was to develop simple, novel and economical UV spectrophotometric methods for determination of Tramadol hydrochloride in bulk and in the form of capsules and tablets, and to validate them in accordance with the protocol for the validation of a method of analysis in chemistry^13,14,15.

 

Fig. 1: Chemical structure of Tramadol Hydrochloride

(Red: O; White: H; Green: Cl; Blue: N; Cyan: C/-CH₃/-CH₂)

 

2. METHODOLOGY:

At first 10mg of Tramadol hydrochloride (Figure 1) was dissolved in 1M of Hydrochloric acid prepared from 37% of Hydrochloric acid because this one is contained in stomach¹⁶;  to find the location of λ_max (Figure 2) witch will be selected for the following analytical wavelength, working standard solution of Tramadol Hydrochloride (Figure 2). Three different diluents of which Distilled Ethanol, 0.1 M of Hydrochloric acid carefully prepared from of Hydrochloric acid (37%, others Features) and distilled water were prepared (Figure 3). A powder equivalent of 10mg was transferred into a 100mL volumetric flask to have 100µg/ml of each diluents wrapped up by aluminum paper to avoid swallowing the light and dissolved as describe under analytical procedure. Results of the analysis were validated as per Protocol for the validation of an analytical method in chemistry and by recovery studies.

 

2.1.    Experimental Chemicals and Reagents:

The pure sample of Tramadol Hydrochloride® (100mg) i.e. was purchased from pharmacy, the expiry of which was not less than 2 year at the time of study. The hydrochloric Acid and Absolute Ethanol were purchased from VWR.

 

2.2.    Instrumentation:

Electrical analytical balance (Precisa 205 A Certified ISO 9001). UV visible spectrophotometer (Model EVOLUTION 300 UV-VIS) connected to HP computer (Compaq LE 2001 W home edition) loaded with its printer (HP laser jet P 1102) with a fixed bandwidth (2nm) and two of 1-cm quartz cell was used for Spectral and absorbance measurements was used in these studies. A Distiller (wassermangels icherung 2001/4), used for distillation of H₂0. Used software package are: VISIONpro; Microcal Origin 6; Chem Sketch; CHNMR Viewer, 3D Viewer. 

 

2.3. Validation of proposed method:

2.3.1. Linearity:

From std. stock solutions of Tramadol Hydrochloride (1000µg/ml), pipette out aliquots of 0.1 to 12ml of Tramadol Hydrochloride transferred to series of 100ml volumetric flasks and final volume made up to mark with distilled water as diluent to form solutions of 1 to 120 µg/ml of Tramadol Hydrochloride. These solutions were then scanned in the range from 200 to 400nm against diluent as blank at λmax of Tramadol Hydrochloride and finally calibration curve was plotted as absorbance vs. concentration to check the linear relationship between absorbance and concentration of Tramadol Hydrochloride. Acceptance Criteria: Correlation Coefficient should be greater than 0.995.

 

2.3.2. Precision:

Precision study expressed by carrying out Repeatability (intraday and Interday precision). They were carried out by estimating corresponding responses three times on the same day and on the three different days for the concentration for (100µg/ml) for Tramadol Hydrochloride. The results of precision study were reported in terms of percentage relative standard deviation. Acceptance Criteria: The Relative Standard Deviation should not be more than 2%. 

 

2.3.3. Accuracy:

The accuracy of developed method was carried out by calculating the % recovery of Tramadol Hydrochloride by standard addition method at three different levels i.e. 80 %, 100% and 120% which are equivalent to be respectively 80, 100 and 120µg/ml of standard solutions of Tramadol Hydrochloride.

 

2.3.4. Limit of Detection and Limit of Quantification:

Limit of detection (LOD) is the lowest concentration of analyte that can be detected while limit of quantitation is defined as lowest concentration of analyte that can be quantitated. With suitable precision and linearity. LOD and LOQ can be calculated from the following formulas: LOD = 3* σ and LOQ = 10 * σ   Where σ is the Standard deviation of the response¹⁷.

 

2.3.5. Ratio of similarity:

The Ratio of similarity allowed indicating the validity of introducing the limit of detection.

 

Ratio of similarity can be calculated using following experimental procedure for the validation of analysis method in chemistry guidelines.:  Where  = average of ten determination and LOD is the limit of detection. Acceptance Criteria: The Ratio of Similarity  should be between 4 and 10¹³.

 

2.3.6. Application on seized materials supposed to contain Tramadol hydrochloride (samples):

2.3.6.1. Sampling:

Thirteen (13) samples for analysis were obtained from OCRTIS (the Central Office for the Suppression of Illegal Traffic in Narcotics) and the court of justice of Niger). The drug samples specialty were purchased from private pharmacies (licensed market). All the thirteen drug samples are obtained with request for authorization addressed to the public prosecutor of Niamey (Niger) and a signature of discharge after receipt of the samples. Each drug sample was then labelled (T_{i = 1 –13}) and put into a plastic bag. The drugs were stored in a fridge in the laboratory (4°C). Their indicated active ingredients (a.i.) contents, weights and origins (OCRTIS or the court of justice) are presented in Table 1¹⁸.

 

Table 1: Tramadol hydrochloride drugs samples characteristics

Sample label	Indicated a.i content	Measured a.i	Obtained form and surname	Origin
T1	225 mg	498.28 mg	Tablet, AAROL-X	OCRTIS
T2	225 mg	615.15 mg	Tablet, Scellé 100/2018	Court of justice
T3	100 mg	386.41 mg	Tablet, CEAGRADOL	OCRTIS
T4	100 mg	390.1 mg	Tablet, Scellé 99/2017	Court of justice
T5	225 mg	302.19 mg	Tablet, Scellé 102/2018,	Court of justice
T6	200 mg	314.85 mg	Tablet, T-Royal,	OCRTIS
T7	200 mg	320.61mg	Tablet, TAMRAL	OCRTIS
T8	200 mg	243.67 mg	capsule, TRAMADOL,	OCRTIS
T9	120 mg	140.66 mg	capsule, Scellé 97/2017	Court of justice
T10	120 mg	149.13 mg	capsule, TRAMADOL	OCRTIS
T11	225 mg	349.27 mg	Tablet, ROYAL,	OCRTIS
T12	225 mg	303.13 mg	Tablet, TAMRAL	OCRTIS
T13	225 mg	542.22 mg	Tablet, Scellé 103/2018	Court of justice

T: Tramadol, a.i.: active ingredient

 

For each drug, tablets or capsules were weighted, average weight calculated and triturated to fine powder and then weight equivalent 100mg of Tramadol hydrochloride  were transferred to 100ml of volumetric flask containing proposed diluent (distilled water); protected from light with aluminum paper to avoid degradation of their active ingredient before analysis, then sonicated 15 minutes and filtered through Whatman filter paper of Tramadol hydrochloride. Pipette 1ml of each solution and transfer it in another 100ml of volumetric flask in the same conditions; so the final concentration will be 10µg/ml. For each drug, three replicate determinations were done and the average of absorbance was taken for the calculating a.i. content. The sample corresponding concentration was calculated by using the calibration curve.

 

2.3.6.2. Determination and analysis of active ingredient content for specialty and samples:

All a.i. (Active ingredient) of samples were estimated in seized trafficking capsules and tablets with label claim from 100 to 225mg/capsule or tablet. The contents of 10 capsules or tablets were weighed and their contents were finely powdered. The average weight of 10 capsules or tablets were also determined. An accurately weighed quantity oh powder equivalent to 100mg oh Tramadol hydrochloride were taken into volumetric flask (100mL) with distilled water then sonicated 15 minutes and filtered through Whatman filter paper like mother stock solution. pipette out aliquots of 1ml of Tramadol Hydrochloride transferred to other 100ml volumetric flasks and final concentration will be 10µg/ml with distilled water as diluent. The absorbance of each the solution was measured in cell against solvent blank at 271nm. For each sample, all the analysis were repeated for tree times and then the average absorbance was taken and the active ingredient of tramadol hydrochloride was calculated by using the following methods equation (A and B)^14,18.

 

 ) ×100% (Method equation A),

where

T: Active ingredient content in percent

DO: Optical Density or Absorbance

E: Molar Extinction Coefficient

E _{1 %} = 1g/100 ml = 1000mg/100ml = 10mg/ml = 10,000 μg/ml

C_f.: Final concentration (10ˠ = 10μg/mL)

 

) ×100% (Method equation B),

 

where

DO_èch: Absorbance of the sample;

DO_rèf: Absorbance of the reference.

 

3.  RESULTS AND DISCUSSION METHOD DEVELOPMENT AND OPTIMIZATION:

3.1. Location of λ_max:

The selection of analytical wavelength, working standard solution of Tramadol Hydrochloride was scanned in the spectrum mode from 200 to 400nm separately in 1.0cm quartz cell against solvent blank. From the UV spectra of drug, λmax of Tramadol Hydrochloride selected for the estimation of drug show 271nm for the analysis (Figure 2)^{3, 8, 20}.

 

Fig. 2: Determination of the Absorption maxima of Tramadol Hydrochloride

 

3.2. Preliminary solubility studies of drug:

10 mg of Tramadol Hydrochloride was weighed and solubility was checked in 100mL of distilled water, ethanol, and 0.1 M HCl. The drug was found to be freely soluble in ethanol with the highest peak and practically poorly soluble in water, and 0.1 M HCl (Figure 3). All diluents give good peak. The highest peak was found with the ethanol. (Figure 3). Then water was selected as diluent in this study because it is the least coast diluent and it could de ingested with water^3,16,19,20.

 

Fig. 3 : Absorption Spectrum of  Ttamadol in différent diluents

 

Then the best time that Tramadol Hydrochloride gives the highest peak has been measured from 0 to 90 minutes. The best peak was found to be at 20 minutes. After this time all the peak fall. This change of concentration was probably the consequence of ligth obsorption. It is ullustrated by the Figure 4.

 

Fig. 4: Stability time of  Ttamadol in distilled water

 

3.3. Preparation of standard stock solutions:

Standard stock solution of Tramadol (1000μg mL^-1) was prepared by weighing 100mg of Tramadol and transferred to a 100mL volumetric flask and volume was made up to 100mL with distilled water to get a concentration of 1000μg mL^-1, the prepared solution is wrapped up by aluminum paper to avoid swallowing the light. Then sonicated for 15 minutes and final volume and filtered through the Whatman filter paper to obtain 1000µg/ml form standard stock solution of Tramadol Hydrochloride.

 

1.4.    Validation:

1.4.1.    Preparation of calibration curve:

From above working std. stock solution of Tramadol Hydrochloride (1000µg/ml), some appropriate volumes of this solution were further diluted to obtained final concentrations in range of 1 to 120μg mL^-1 (table 1). The spectrum of this solution was recorded in the range of  200 - 400 nm. These solutions were then scanned in the range of 200-400nm against diluent as blank and then calibration curve was plotted as absorbance vs. concentration.

 

1.4.2.    Linearity:

Linearity was evaluated by analysis of Std. Tramadol Hydrochloride at eleven different concentrations. Tramadol Hydrochloride found to be linear within conc. range of 1-120µg/ml with regression coefficient of 0.9996. The results of regression analysis are summarized in (Table 2). Results shows that there was an excellent correlation between peak area and concentration. (Figure 5)^{3, 7, 20}.

 

Table 2: Data curve calibration

Sr. N°	Concentration in µgmL-1	Wavelength	Absorbance	R2
1	1	271 nm	0,0095	0,9996
2	8		0,054
3	10		0,07
4	12		0,083
5	20		0,137
6	40		0,258
7	60d		0,381
8	80		0,506
9	90		0,56
10	100		0,619
11	120		0,734

 

Fig. 5: Calibration Curve of Tramadol hydrochloride

 

1.4.3.    Precision:

The precision of an analytical method is determined by assaying ten determinations at test concentration (10µg mL^-1). Percentage of Relative Standard Deviation (% RSD) calculates statistically (Table 3). It was found to be less than 2% (0,731 %) indicate the high precision of the propose method. The precision of an analytical method is determined by assaying ten determinations at test concentration (10 µg mL^-1)^{8, 21}.

 

Table 3: Precision Data of the proposed method

 

Table 4: Precision Data of the proposed method for the lowest concentration

C_R: real concentration C_S: sample concentration, Abs.: absorbance, LOD: limit of detection, LOQ: limit of quantification.

 

Intraday and Interday precision:

The repeatability (intra-days precision) is expressed as percentage relative standard deviations (% RSD). The average of the percentage of RSD value of intra-days precision for Tramadol Hydrochloride at the concentration of 100µg/ml were 0.7804 and 0.5398 for inter-days precision. In all cases, the percentage RSD were less than 2. So there is no significant variations in the analysis of Tramadol Hydrochloride at the concentrations and the proposed method was precise which are shown in Tables 5 and 6^3,21.

 

Table 5: Results of Intraday Precision Study

Abs.: absorbance

 

Table 6: Results of Interday Precision Study

Abs.: absorbance

 

3.4.5.    Accuracy (Recovery Study)

The accuracy was assessed by the standard addition method of three replicate determinations of three different solutions containing 8, 10 and 12 µg mL^-1 of Tramadol Hydrochloride. The average % recoveries for three different concentrations were found to be 100,862 % using proposed UV spectrophotometric method. The higher values indicated that the proposed UV spectrophotometric method was accurate for the determination of Tramadol Hydrochloride in pharmaceutical dosage form. All the results of recovery studies are shown in Table 7^{3, 5, 7, 19, 21, 22}.

 

3.4.6.         Limit of Detection and Limit of Quantification:

The value of the limit of detection was found to be 0.177 µg mL^-1 and the limit of quantification was found to be 0.592 µg mL^-1. This indicates that the developed method was sensitive for the estimation of Tramadol Hydrochloride in bulk, capsules and tablet dosage form. The results of LOD and LOQ are shown in Table 3^3,17.

 

3.4.7.    Ratio of similarity:

The Ratio of similarity was found to be 5.71. This value means the concentration used is appropriate for the determination of results and analytical methodology. The result of The Ratio of Similarity is shown in Table 4¹³.

 

3.5. Results of specialty and samples analysis:

3.5.1. Tramadol hydrochloride specialty:

Twenty capsules containing 100 mg of Tramadol Hydrochloride weighed, average weight calculated and triturated to fine powder and then weight equivalent 100 mg of Tramadol hydrochloride were transferred to 100ml of volumetric flask containing proposed diluent, then sonicated 15 minutes and filtered through Whatman filter paper of Tramadol hydrochloride like std. stock solution. The present UV spectrophotometric methods was validated for linearity, precision, accuracy, LOD and LOQ using following experimental procedure for the validation of analysis method in chemistry guidelines (Canada). Acceptance Criteria: Mean recovery should be in the range of 99-101 %.^8,15

 

Table 7: Method Accuracy from Recovery Assay

Sr. No°	Sample identity	[ ] en µgmL-1	Absorbance	Absorbance average	Amount Recovered in µgmL-1	% Recovered	% Recovered
1	Accuracy 80 %	8	0,053	0,0546	8,088	101,234	100,862
		8	0,056
		8	0,055
2	Accuracy 100 %	10	0,071	0,0706	10,085	100,952
		10	0,070
		10	0,071
3	Accuracy 120 %	12	0,084	0,0833	12,043	100,401
		12	0,083
		12	0,083

 

3.5.2. Samples vs specialty analysis:

Thirteen tramadol drugs were analyzed in this study. Those samples drugs were obtained from the Central Office of damaging Trafficking drugs (OCRTIS) (61.5 %) and court of justice (38.5 %) of Niamey.

 

Among the thirteen drugs, T₁,T₃ ,T₉ from OCRTIS and respectively T₂, T₄ , T₁₀ from the court of justice have the same indicated name, manufacturer and distributor. The active ingredient (a.i) contents of the specialty is calculated by using method equation A. the active ingredient contents of the drugs samples were calculated by using the following method equation B. They are summarized in Table 8.

 

Table 8: results of the a.i. percent of the specialty and seized materials containing tramadol

Sample label	Abs.	Method B (% samples)	Pharmaceutical Product (%)	Method A  (% specialty)
T1	0.056	80	T	100
T2	0.041	58.57
T3	0.041	58.57
T4	0.027	38.57
T5	0.063	90
T6	0.029	41.43
T7	0.054	77.14
T8	0.071	101.43
T9	0.079	112.86
T10	0.068	97.14
T11	0.066	92.28
T12	0.064	91.43
T13	0.062	88.57

T: Tramadol, Abs.: absorbance

 

According to the results, all the thirteen-(13) tramadol hydrochloride drugs obtained from OCRTIS and the court of justice with irregular dosage and market have substandard concentrations when compared to the specialty claimed content (100mg a.i). All the samples are fake drug; they are not conforming to manufacturing standards (99 -101%). They contains less than 99 % or more than 101% claimed a.i content. Among all the samples products obtained from licensed market, sample T₈ (101.43% of the indicated a.i) is the best; it is a relatively good drug; T₉ have very high standard concentrations. All the rest have substandard, they contain less than 99% of the indicated a.i. The presence of substandard drugs in seized materials estimated containing tramadol hydrochloride is probably due to the infiltration of the official market by substandard and fake medicaments.

 

Comparing the results obtained T₈; the relatively good drug in the seized materials supposed to contain tramadol hydrochloride shows that, this product is probably not a counterfeit. Indeed, real licensed drugs also infiltrate the illicit market. T₅, T₁₀, T₁₁ and T₁₂ which active ingredient are ≥ 90 %; is probably due to the bad conservation conditions of this drug because it is easy to hide them in counterfeiting medications. This is one of the reason of drug poor quality. The active ingredient decomposition under bad conservation conditions was occasionally the cause of drugs poor quality in least developed countries. The infiltration of fake and substandard medicines in the official circuit is generally due to the absence of regular quality control mechanisms.

 

4.  CONCLUSION:

A fast, non-toxic, reasonably priced and simple method for the determination of Tramadol Hydrochloride in bulk, capsules and tablet dosage have been developed and validated by using the UV spectrophotometer. The results of the validation parameters show that the UV spectrophotometric methods were accurate, precise and sensitive. It is reproducible in this range and can be used in case of possible Tramadol seizures or quality controls of this drug. This method can be used in laboratories for the quantitative analysis of Tramadol Hydrochloride in pharmaceutical formulations for quality control, where economy and time are essential to give good results. The present study showed us the important presence of substandard and fake seized materials containing tramadol hydrochloride drugs in seized materials. In the thirteen samples, eleven have a substandard concentrations; one is a relatively good drug (T₈) and T₉ have a.i. >101 %. According to the seizure of these drugs and their related potential health risks, it’s emergency to sensitize youngers about drugs. It is also important to make their quality control in the country because these drugs can be used in scientific research and in hospital for needy persons before incinerating them like the laws of court require.

 

5.    ACKNOWLEDGEMENTS:

The authors are very thankful to:

The National Gendarmerie of Niamey,

The University (U A M) of Niamey;

The Central Office of damaging Trafficking drugs (OCRTIS) of Niger;

The Law Ministry of Niamey;

The public prosecutor at a tribunal de Grande instance (TGI/HS) of Niamey.

 

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Received on 11.12.2019                    Modified on 07.01.2020

Accepted on 29.01.2020                   ©AJRC All right reserved