INTRODUCTION:

Chlorzoxazone is a “Skeletal muscle relaxant” type of drug with several targets, it inhibits “multisynaptic reflex involved in producing and maintaining skeletal muscle spasm of varied etiology. Abdominal pain or tenderness clay-coloured stools, dark urine, decreased appetite, fever, headache, itching, nausea, vomiting, skin rash, swelling of the feet’s and lower legs." are other actions linked to chlorzoxazone use².^3,4

The Chemical name of this drug is “5-chloro-2-benzoxazolinone”⁵

The powder form of chlorzoxazone is white in color. This drug is slightly soluble in water, and sparingly soluble in alcohol, isopropyl alcohol and methanol and practically odorless powder.^6,7 There are oral capsules preparations available in tablet and capsule form.⁸

For the management of certain skeletal muscle relaxation, chlorzoxazone oral dose is 500 mg three or four times a day.

In individuals suffering from chronic painful muscle⁹ the mechanism of action of chlorzoxazone is to inhibit degranulation of mast cells, subsequently preventing the release of histamine and slow-reacting substance of anaphylaxis (SRS-A), mediators of type I allergic reactions. Chlorzoxazone also may reduce the release of inflammatory leukotrienes. Chlorzoxazone may act by inhibiting calcium and potassium influx which would lead to neuronal inhibition and muscle relaxation. Data available from animal experiments as well as human study indicate that chlorzoxazone acts primarily at the level of the spinal cord and subcortical areas of the brain where it inhibits multisynaptic reflex arcs involved in producing and maintaining skeletal muscle spasm .¹⁰

Primary goal of this study was to locate an appropriate method of analysis by UV-Spectrophotometer. The solubility of the drug chlorzoxazone was studied and compared in-between three different types of body fluids with three different pHs those are physiological buffer, stomach buffer and intestinal buffer. The three different types of buffers represent the three different body solutions. The best suitable solution was found out and the best suitable site for administration was determined.

MATERIALS AND METHODS:

Materials:

Chlorzoxazone was procured from CIPT College. Sodium chloride, Sodium dihydrogen phosphate and di-sodium nitrogen phosphate and potassium chloride, hydrochloric acid, pepsin, sodium hydroxide, calcium chloride, magnesium chloride, and sodium bicarbonate were given by CIPT College. The grade of all other chemicals utilized was analytical grade.

Spectrophotometric analysis was done with a double beam UV visible Spectrophotometer with a 10 millimeters path length in quartz cell, for analytical purpose¹¹.

Preparation of physiological simulator buffer solution (7.4 pH):

0.8 g of NaCl was precisely weighed, then dissolved into 10 ml distilled water. Sodium di-hydrogen phosphate, di-sodium hydrogen phosphate and KCl were accurately weighed and put into the solution¹². And the volume of solution contained up-to 100 millilitres of distilled water. Then the pH 7.4 was adjusted. This 100 ml solution was applied as the diluent^13,14.

Preparation a standard curve of chlorzoxazone in physiological solution (pH 7.4)

10 milligrams of the drug was diluted into ten millilitres of the buffer solution to get 1mg per ml or 1000 mcg/ml stock solution¹⁵. Then further serial dilutions were done with the previously made buffer, to prepare the concentration range of 5, 10, 15, 20, 25, 30 and 35 μg/ml. And the absorption was determined using a UV spectrophotometer at 232 nm¹⁶ against a blank of pH 7.4 phosphate buffer. The standard calibration curve was obtained by plotting the absorbance with concentration (μg/mL).

Preparation of gastric simulator buffer solution (1.2 pH):

0.2 gm of NaCl was weighed accurately and dissolved into 0.7 ml of HCl. And 0.32g of pepsin was precisely measured and dissolved in 10 ml of distilled water¹⁷ and the volume was filled up to 100 ml by pouring more distilled water. Then the PH was maintained to 1.2 by adding HCl¹⁸. This 100 ml solution was used as the diluent.

Preparation of standard curve of chlorzoxazone in gastric simulator buffer solution (pH 1.2):

Ten milligrams of the drug was diluted into 10 ml of the buffer solution to get 1mg/ml or 1000 mcg/ml stock solution¹⁹. Then further serial dilutions were done to prepare the concentration range of 5,10,15,20,25,30 and 35 μg/ml. And the absorption was evaluated in comparison to pH 1.2 buffer as the blank at utilizing a UV spectrophotometer, 232 nm²⁰. The “standard calibration curve” was obtained by plotting the absorbance with concentration (μg/mL).

Preparation of intestine simulator buffer solution (6.8 pH)

0.1 gm of NaOH, 1.19 gm NaCl, 0.1g of KCl, 0.04 gm of CaCl₂, 0.02 gm MgCl_, 0.2 gm of sodium bicarbonate were weighed accurately, and dissolved into 10 ml of water and then a 100 ml amount was added which was distilled water²¹. Then The pH was changed to 6.8. This 100 ml solution was applied as the diluent.

Preparation of standard curve of chlorzoxazone in intestinal solution (pH 6.8)

10 mg of the drug was diluted into 10 ml of the buffer solution to get 1mg/ml or 1000 μg/mL stock solution²². Then further serial dilutions were done to have the concentration range of 5, 10, 15, 20, 25, 30 and 35 μg/ml. And absorption was measured against pH 6.8 buffer as the blank at 232 nm using a UV-spectrophotometer²³. The “standard calibration curve” was created by plotting the absorbance versus the concentration (μg/mL).

RESULT:

The purpose of this investigation was to determine the best appropriate solution for developing a simple and precise UV-spectrophotometric method and to select a suitable absorption site for chlorzoxazone. This investigation includes figuring out the concentration of the drug in three types of body fluids spectrophotometrically.

The all three concentrations against absorbance standard curve graphs, determined by UV spectrophotometer, of the drug chlorzoxazone, in three different buffer media was observed and comparison was done in-between them. The simulator fluid which could stabilize the region with the highest drug concentration was chosen to be the focus of the administration of adjusted dose forms of chlorzoxazone. Findings and discussions of the above study is presented below.

Table 1: Calibration curve readings of chlorzoxazone in pH 7.4 buffer at 232 nm

Concentration (mcg/ml)	Absorbance at 232 nm
0	0
5	0.11
10	0.18
15	0.250
20	0.31
25	0.397
30	0.45
35	0.518

Figure 1: Standard curve of chlorzoxazone in pH 7.4 buffer at 232 nm.

Table 2: Calibration curve readings of chlorzoxazone in pH 1.2 buffer at 232 nm

Concentration (mcg/ml)	Absorbance at 223 nm
0	0
5	0.05
10	0.130
15	0.199
20	0.246
25	0.301
30	0.360
35	0.418

Figure 2: Standard curve of chlorzoxazone in pH 1.2 buffer at 232 nm.

Table 3: Calibration curve readings of chlorzoxazone in pH 6.8 buffer at 232 nm

Concentration (mcg/ml)	Absorbance at 232 nm
0	0
5	0.145
10	0.238
15	0.372
20	0.468
25	0.592
30	0.692
35	0.818

Figure 3: Standard curve of chlorzoxazone in pH 6.8 buffer at 232 nm.

Table 4: Comparison between calibration curve of chlorzoxazone, in three body fluids, at 232 nm

Concentration (mcg/ml)	Physiological solution (7.4 pH)	Gastric solution (1.2pH)	Intestinal solution (6.8pH)
0	0	0	0
5	0.11	0.05	0.145
10	0.18	0.130	0.238
15	0.250	0.199	0.372
20	0.31	0.246	0.468
25	0.397	0.301	0.592
30	0.45	0.360	0.692
35	0.518	0.418	0.818

Figure 4: Comparison between Standard curves of chlorzoxazone, in three body fluids, at 232 nm

From the above comparison between three different pH buffers, we can see that the chlorzoxazone drug is mostly soluble in 6.8 pH phosphate buffer solution.

Sensitivity:

The formula LOD= 3*σ/ S and LOQ= 10 * σ/ S were utilized for calculating the limits of detection and limit of quantification, respectively. Here sigma signifies the standard deviation of intercept and S is referring the slope. The LOD of gastric fluid and intestinal fluid and physiological It was discovered that fluid 0.3968 µg/ml, 0.375 µg/ml and 0.3158 µg/ml respectively. The LOQ of gastric fluid intestinal fluid and physiological It was discovered that fluid was 1.2025 µg/ml, 1.30 µg/ml and 1.9832 µg/ml respectively.

DISCUSSION:

According to figure 4, from the comparison between three different pH buffers, we can conclude that the chlorzoxazone drug is mostly soluble in 6.8 pH phosphate buffer solution.

Figure 4 displays the solubility of chlorzoxazone in different body fluids. It was discovered through solubility research that exhibited the maximum solubility in the intestinal fluid. And in the physiological and gastric envioronment, solubility is intermediate and lowest respectively.

So, the most suitable buffer for UV Spectrophotometric analysis of the drug chlorzoxazone would be the intestinal buffer of pH 6.8.

CONCLUSION:

The primary goal of the research was to design the most suitable UV-spectrophotometric determination method and to ascertain the body's appropriate absorption site for the creation of an appropriate adjusted dosage form for the medication chlorzoxazone. The comparison of three different body fluids solubility of the drug was done. The drug in each body fluid was assessed using spectrophotometry. The results of UV Spectrophotometry analysis verified that there was a higher drug concentration in the intestinal fluid, compared to physiological fluid and gastric fluid. Therefore, the formulation would be changed in a manner that would dosage form will deliver the maximum drug into the intestinal fluid or the small intestine.

CONFLICT OF INTEREST:

The authors not having any conflicts of interest regarding this thorough investigation.

ACKNOWLEDGMENTS:

The authors are thanking CIPT and AHS college authority for their kind support during all lab studies.

REFERENCES:

1. Ravisankar P, Anusha S, Babu PS. Development and validation of UV-spectrophotometric method for determination of dasatinib in bulk and pharmaceutical dosage form and its degradation behaviour under various stress conditions. Int. J. Pharm. Sci. Rev. Res. 2020; 53: 45-50.

2. Nuli MV, Rekulapally VK. Overall review on analytical method development and validation of Dasatinib. International Journal of Pharmacognosy and Chemistry. 2023; 27-34.

3. Mittha J, Habib B. UV spectrophotometric method development and validation of dasatinib in bulk and formulation. Asian Journal of Pharmaceutical Analysis. 2021; 11(3): 203-6.

4. Bayas JP, Sumithra M. Analytical method development and validation of Dasatinib in Bulk and pharmaceutical formulation using quality by design. Research Journal of Pharmacy and Technology. 2021; 14(3): 1591-6.

5. Sultana M, Rizwana DI. Stability indicating method development and validation for simultaneous estimation of dasatinib and lenvatinib by using uplc in pharmaceutical dosage form. World J Pharm. 2020; 9: 1548-60.

6. Shi M, Wang L, Li P, Liu J, Chen L, Xu D. Dasatinib–SIK2 binding elucidated by homology modeling, molecular docking, and dynamics simulations. ACS Omega. 2021; 6(16): 11025-38.

7. Reddy A, Parthiban S, Vikneswari A, Senthilkumar G. A modern review on solid lipid nanoparticles as novel controlled drug delivery system. Int. J. Res. Pharm. Nano Sci. 2014; 3(4): 313-25.

8. Müller RH, Mäder K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drug delivery–a review of the state of the art. European Journal of Pharmaceutics and Biopharmaceutics. 2000; 50(1): 161-77.

9. Trevaskis NL, Charman WN, Porter CJ. Lipid-based delivery systems and intestinal lymphatic drug transport: a mechanistic update. Advanced Drug Delivery Reviews. 2008; 60(6):702-16.

10. Yadav P, Soni G, Mahor A, Alok S, Singh PP, Verma A. Solid lipid nanoparticles: An effective and promising drug delivery system-A review. International Journal of Pharmaceutical Sciences and Research. 2014; 5(4): 1152.

11. Rathod SD, Patil PM, Jadhav SB, Chaudhari PD. UV spectrophotometric simultaneous determination of metformine hydrochloride and pioglitazone hydrochloride in combined dosage form. Asian Journal of Pharmaceutical Analysis. 2012; 2(1): 5-9

12. Madhu C, Swapna J, Spandana RS, Niharika I, Raj KR, Kalyan B, Rajarajeshwari M, Prathyusha T. Quantitative evaluation of carbohydrate levels in seeds for home use by UV-visible spectrophotometer. Asian Journal of Pharmacy and Technology. 2012; 2(3): 110-1.

13. Sourav Duari, Ayan Das, R.N. Pal. UV-Spectrophotometric Method Development and Determination of Suitable Medium for Lornoxicam by Comparing In-Between Three Body Fluids. Asian Journal of Pharmaceutical Analysis. 2025; 15(2): 75-9.

14. Ankit Sharma, S. K. Arora, Manoj Kumar Batra, Rakhi Khandelwal. Corrosion inhibition and Adsorption characteristics of 3,4,5-trihydroxy-n-(3,4-dimethoxy benzylidene) benzo hydrazide schiff base on aluminium in different concentration of Hydrochloric acid environment. Research Journal of Science and Technology. 2023; 15(4): 175-2.

15. S. B. Gondkar, Nikita S. Malekar, R.B. Saudagar. An Overview on Trends and Development of Niosomes as Drug Delivery. Research J. Topical and Cosmetic Sci. 2016; 7(2): 79-85.

16. Shabina Fatma, Kiran Kumari. Study of Narcotic substances present in different Biological samples. Research Journal of Science and Technology. 2023; 15(4): 203-4.

17. Tomasz W. Kamiński, Małgorzata Karbowska, Dariusz Pawlak. A Review of the Pharmacological properties of potential drugs for the treatment of stuttering from the past to the future. Asian J. Pharm. Res. 2018; 8(2): 104-109.

18. Manoj Kumar Ghosh, Harsha Tiwari. Heavy Metal Concentration in underground water due to Fly Ash at Janjgir Champa region in Chhattisgarh. Research Journal of Science and Technology. 2021; 13(2): 79-4.

19. S. D. Mankar, Sahil B. Shaikh. Microencapsulation: is an advance Technique of Drug formulation for Novel Drug Delivery System. Research J. Science and Tech. 2020; 12(3): 201-210.

20. Jyoti Prakash, Shweta Manan, Vinod Kumar. On Triply Diffusive Convection in Porous Medium: Darcy Brinkman Model. Research J. Science and Tech. 2017; 9(1):127-130.

21. Jyoti Prakash, Rajeev Kuma, Prakash Chopra. On Triply Diffusive Convection Analogous to Stern type with Variable Viscosity. Research J. Science and Tech. 2017; 9(1): 111-114.

22. Manish Upadhyay. Depleting Groundwater Levels and Increasing Fluoride Concentration in Villages of Surajpur District, Chhattisgarh, India: Cost to Economy and Health. Research J. Science and Tech. 2012; 4(3): 122-128.

23. Shyam S Kumar, G. Gopalakrishnan, N. L. Gowrishankar. Design, Optimization and in vitro Characterization of Dasatinib loaded PLGA Nano carrier for Targeted cancer therapy: A Preliminary Evaluation. Research Journal of Pharmacy and Technology. 2021; 14(4): 2095-0.

Received on 05.05.2025 Revised on 30.06.2025

Accepted on 07.08.2025 Published on 08.10.2025

Available online from October 15, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(4):314-318.

DOI: 10.52711/2231-5675.2025.00049

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