Development and Validation of UV Spectrophotometric and RP-HPLC Methods for Quantitative Estimation of Levosalbutamol Sulphate in Pharmaceutical Dosage Forms

Levosalbutamol sulphate is the pharmacologically active R-enantiomer of salbutamol and is widely used as a selective β2-adrenergic receptor agonist for the treatment of bronchial asthma and chronic obstructive pulmonary disease (COPD). It produces bronchodilation by relaxing bronchial smooth muscles and provides rapid relief from airway obstruction. Compared with racemic salbutamol, levosalbutamol exhibits improved therapeutic efficacy and fewer cardiovascular adverse effects because it lacks the inactive S-enantiomer, which has been associated with undesirable pharmacological responses.¹^-²

Analytical quality control plays an essential role in ensuring the safety, efficacy, and consistency of pharmaceutical products. Reliable analytical methods are required for routine assay, dissolution testing, stability studies, and quality assurance during manufacturing. The selected analytical method should provide high accuracy, precision, specificity, sensitivity, and reproducibility while remaining economical and simple to perform.³ Among the available analytical techniques, UV-visible spectrophotometry is one of the most commonly employed methods because of its simplicity, rapid analysis, and cost-effectiveness. It is extensively used for routine quantitative estimation of pharmaceutical compounds. However, UV spectroscopy has limitations in selectivity when formulation excipients or degradation products interfere with drug estimation. Therefore, chromatographic techniques such as Reverse Phase High Performance Liquid Chromatography (RP-HPLC) are preferred for pharmaceutical analysis requiring higher sensitivity and specificity.⁴

RP-HPLC has become the analytical technique of choice in pharmaceutical industries because of its excellent resolution, reproducibility, short analysis time, and capability to separate analytes from impurities and degradation products. Method optimization involves selecting an appropriate stationary phase, mobile phase composition, flow rate, detection wavelength, and sample preparation conditions to achieve efficient chromatographic separation.⁵ Before an analytical method can be routinely applied, it must undergo systematic validation to demonstrate its suitability for the intended purpose. According to the International Council for Harmonisation (ICH) guideline Q2(R2), analytical methods should be validated for specificity, linearity, accuracy, precision, robustness, limit of detection (LOD), limit of quantification (LOQ), and range. Proper validation ensures that the developed method consistently produces reliable and reproducible analytical results.⁶ Several analytical methods have been reported for the estimation of bronchodilator drugs; however, there remains a need for a simple, rapid, economical, and fully validated method suitable for routine quality control laboratories.

The present investigation aimed to develop and validate both UV spectrophotometric and RP-HPLC methods for the quantitative estimation of Levosalbutamol sulphate in pharmaceutical dosage forms according to current ICH guidelines. The developed methods are expected to provide reliable analytical performance for routine pharmaceutical quality control and regulatory applications.

MATERIALS AND METHODS

Materials

Levosalbutamol sulphate reference standard (purity ≥99.0%) was obtained from a certified pharmaceutical manufacturer. Commercial tablet formulations containing Levosalbutamol sulphate were procured from the local market. HPLC-grade acetonitrile, methanol, orthophosphoric acid, HPLC-grade water, and analytical-grade chemicals were used throughout the study. All reagents complied with the specifications of the Indian Pharmacopoeia (IP) and were used without further purification. Glassware and volumetric apparatus were calibrated before use.⁸

Instrumentation

UV spectrophotometric analysis was carried out using a double-beam UV–Visible spectrophotometer equipped with 1 cm quartz cells. RP-HPLC analysis was performed using a high-performance liquid chromatographic system equipped with a quaternary pump, autosampler, PDA detector, and chromatography software. Separation was achieved using a Phenomenex Kinetex XB-C18 column (150 × 4.6 mm, 5 μm).⁹

UV Spectrophotometric Method

A standard stock solution of Levosalbutamol sulphate was prepared in methanol. The solution was scanned between 190–400 nm, and the maximum absorbance (λmax) was found at 226 nm. Working standard solutions were prepared in the concentration range of 40–60 μg/mL. The absorbance of each solution was measured at 226 nm, and a calibration curve was constructed by plotting absorbance against concentration. The developed method was validated for linearity, accuracy, precision, repeatability, LOD, and LOQ according to ICH Q2(R2) recommendations.¹⁰

RP-HPLC Method Development

Chromatographic separation was achieved using a Phenomenex Kinetex XB-C18 column (150 × 4.6 mm, 5 μm). The optimized mobile phase consisted of 0.1% orthophosphoric acid and acetonitrile (30:70, v/v), delivered at a flow rate of 1.0 mL/min. The injection volume was 10 μL, the detection wavelength was 226 nm, and the total run time was 10 min. Before use, the mobile phase was filtered through a 0.45 μm membrane filter and degassed by ultrasonication. Standard and sample solutions were prepared using the optimized diluent and filtered before injection into the HPLC system.¹¹

Method Validation

The developed UV spectrophotometric and RP-HPLC methods were validated in accordance with ICH Q2(R2) guidelines. Validation parameters included specificity, linearity, accuracy, precision, repeatability, robustness, limit of detection (LOD), and limit of quantification (LOQ). Linearity was evaluated over the concentration range of 80–120% of the working concentration. Accuracy was determined by recovery studies at 80%, 100%, and 120% concentration levels. Precision was assessed by intraday and interday studies, while robustness was evaluated by small deliberate variations in wavelength and column temperature.¹²

Statistical Analysis

Experimental results were expressed as mean ± standard deviation. Calibration curves were generated by linear regression analysis, and the correlation coefficient (R²) was calculated to evaluate linearity. Statistical parameters including regression equation, standard deviation, relative standard deviation (%RSD), LOD, and LOQ were calculated using Microsoft Excel and chromatography software. Acceptance criteria were evaluated according to ICH validation requirements.¹³

RESULTS AND DISCUSSION

Organoleptic attributes:

• Color: White to almost white crystalline powder
• Smell (Odor): Odorless
• Taste: Slightly bitter

Physical parameters:

Melting point of Levosalbutamol sulphate: 187–190°C

Solubility:

The solubility of levosalbutamol sulphatewas determined in various solvents as shownin Table 1.

Table No.1 Solubility Chart

UV

Figure 1 UV-Visible absorption spectrum of levosalbutamol sulphate in the selected solvent.

Figure No. 2 UV Spectrum of Levosalbutamol Sulphate showing λmax of 226 nm

1	2
3	4

Fig No 3 Chromatogram of Chromatographic Condition 1,2,3 and 4

80%	90%
100%	110%

Figure No. 4 Chromatograms for Linearity at 80%, 90%, 100%, 110%

120%

Figure No. 5 Chromatograms of Overlay Linearity at 80%, 90%,100%,110%,120%

1	2
3	4
5	6

Figure No. 6 Chromatograms for system suitability of Levosalbutamol

80% Rep 1	80% Rep 2	80% Rep 3
100% Rep 1	100% Rep 2	100% Rep 3
120% Rep 1	120% Rep 2	120% Rep 3

Figure No. 7 Chromatograms for Accuracy at 80%, 100% and 120% level

Table No. 12 Peak Results for Accuracy of Levosalbutamol

Levosalbutamol
Std wt. (mg)	% Purity	Std Stock Conc. (ug/ml)	Working Std Area
5	99.9	49.95	315583

 

Sample ID	Reps	Spiked Conc. (ug/ml)	Area	Amount Recovered (ug/ml)	% Recovery	AVG	STD EV	% RSD
80%	Rep 1	4.00	252765	4.00	100.12	100.02	0.09705	0.10
	Rep 2	4.00	252275	3.99	99.92
	Rep 3	4.00	252515	4.00	100.02
100%	Rep 1	5.00	315951	5.00	100.12	99.96	0.136087	0.14
	Rep 2	5.00	315125	4.99	99.86
	Rep 3	5.00	315334	4.99	99.92
120%	Rep 1	5.99	379894	6.01	100.32	100.21	0.111154	0.11
	Rep 2	5.99	379562	6.01	100.23
	Rep 3	5.99	379058	6.00	100.09

Table No. 13 Robustness Column Temperature 28°C, 30°C, 32°C

Variation in Column temperature
Condition	Sample ID	RT	Area	% Assay
28C	WS	2.28	312467	-
	DP	2.28	311559	99.71
30C	WS	2.28	315951	-
	DP	2.28	314754	99.62
32C	WS	2.28	313254	-
	DP	2.28	312415	99.73
AVG		2.28	313400	99.69
SD		0.00	1648.77	0.06
% RSD		0.00	0.53	0.06

 

280C DP

280C WS

Figure No. 8 Chromatograms for Column temp at 28°C

320C DP

320C WS

Figure No. 9 Chromatograms for Column temp at 32°C

224nm DP

224nm WS

Figure No. 10 Chromatograms for Column Variation in wavelength 224nm

228nm DP

228nm WS

Figure No. 11 Chromatograms for Column Variation in wavelength 228nm

Table No. 14 Robustness Column Variation in wavelength 224nm, 226nm and 228nm

Variation in wavelength
Condition	Sample ID	RT	Area	% Assay
224 nm	WS	2.28	310254	-
	DP	2.28	309112	99.63
226 nm	WS	2.28	315951	-
	DP	2.28	314754	99.62
228 nm	WS	2.28	312121	-
	DP	2.28	311034	99.65
AVG		2.28	312204	99.63
SD		0.00	2656.36	0.02
% RSD		0.00	0.85	0.02

Figure No. 12 Chromatogram of Blank

Figure No. 13 Chromatogram of Levosalbutamol

Figure No.14 Chromatogram of Standard

The developed RP-HPLC method exhibited excellent sensitivity, with LOD and LOQ values of 0.09 µg/mL and 0.26 µg/mL, respectively, confirming its suitability for the detection and quantification of Levosalbutamol sulphate at low concentration levels. LOD and LOQ are indicators of method sensitivity and should not be interpreted as proof of method accuracy, precision, or overall reliability.

CONCLUSION

The present investigation successfully developed and validated UV spectrophotometric and RP-HPLC methods for the quantitative estimation of Levosalbutamol sulphate in pharmaceutical dosage forms. Both analytical methods exhibited excellent linearity, accuracy, precision, specificity, robustness, and sensitivity in accordance with ICH Q2(R2) guidelines. The RP-HPLC method provided efficient chromatographic separation with acceptable retention time and high theoretical plate count, while the UV spectrophotometric method proved to be simple, rapid, and economical for routine laboratory analysis. The low values of LOD and LOQ demonstrated the high sensitivity of the developed methods for detecting and quantifying small amounts of the drug. Overall, the validated analytical methods are reliable, reproducible, and suitable for routine quality control testing, assay determination, and regulatory analysis of Levosalbutamol sulphate in pharmaceutical formulations, thereby ensuring product quality, safety, and therapeutic efficacy.

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