Determination of Spectinomycin by Wayeal High Performance Liquid Chromatograph with ELSD Detector

The Evaporative Light-Scattering Detector (ELSD) is a novel mass detector with theoretical applicability to any compound that can form gaseous particles. The 2025 editionof the Pharmacopoeia specifies the use of Evaporative Light-Scattering Detection (ELSD) for the analysis and quality control of over 100 drug varieties. In practice, ELSD is more frequently applied to compounds lacking ultraviolet (UV) absorption, such as astragaloside, carbohydrates, aminoglycosides, and cholesterol, often serving as a complementary detection method to UV and fluorescence detectors. However, ELSD results are influenced by multiple factors, including mobile phase composition, organic solvent concentration, elution mode, and nebulization conditions. Therefore, to ensure accurate and consistent analytical outcomes, it is essential to evaluate the impact of these factors on the results.
This study, referencing the analytical method for spectinomycin hydrochloride fromPart II of the 2025 Pharmacopoeia and guided by the ICH QbD principles,employed an orthogonal experimental design to screen and optimize the analytical conditions.

Fig 1 Spectinomycin Hydrochloride

2. Instrument and Method

In this study, chromatographic analysis was carried out using Wayeal’s LC3200 series HPLC system. The instrument configuration included a quaternary gradient pump, an autosampler, a column compartment, and an Evaporative Light-Scattering Detector. System control and data acquisition/processing were performed with the SmartLab 2.0 chromatography software.

Table 1 HPLC Conditions

3. Solution Preparation

Sample solution: The stock solution of spectinomycin hydrochloride was provided by the client with a labeled concentration of 10 mg/mL and a purity of 99.8%.

0.1M Trifluoroacetic acid aqueous solution：Precisely pipette 6.8mL of trifluoroacetic acid into a 1000mL volumetric flask. Dilute to the mark with purified water and mix well. Finally, vacuum-filter the solution through a 0.45μm cellulose membrane filter before use.

Standard Solutions: The stock solution was diluted with purified water to prepare sample solutions with concentrations of 0.15, 0.25, 0.35, 0.50, and 0.70mg/mL, respectively. Each solution was then filtered through a 0.45μm cellulose membrane filter before use.

4. Result and Discussion

The Chinese Pharmacopoeia employs identical chromatographic conditions for analyzing related substances and the main component content under the spectinomycin hydrochloride entry. The method states that "the detector can be configured based on actual circumstances." During the study, considering the instrumental characteristics of the ELSD3260, the requirements of the analytical method, and relevant literature, a Plackett-Burman experimental design was employed to screen multiple experimental parameters (Tables 2 and 3). Subsequently, a Box-Behnken design (Table 4) was utilized to optimize the peak area response of the ELSD detector.

Table 2 Plackett-Burman Experimental Design

Table 3 ANOVA Analysis of Plackett-Burman Experimental Results

As can be seen from Table 3, gas flow rate, mobile phase flow rate, and post-column compensation showed negative effects. Specifically, reducing the gas flow rate and mobile phase flow rate, along with disabling post-column compensation, could increase the chromatographic peak response. However, decreasing the mobile phase flow rate may lead to increased peak broadening and reduced detection sensitivity. Therefore, the flow rate was maintained consistent with the standard method. While column temperature and detection temperature exhibited positive effects, they did not significantly enhance the response value. Consequently, subsequent studies will focus on optimizing the gas flow rate, nebulizer temperature, and evaporator temperature.

The effects of gas flow rate, nebulizer temperature, and evaporator temperature on the response value were investigated by employing a Box-Behnken experimental design within a response surface methodology framework. The experimental design is presented in Table 4.

Table 4 Box-Behnken Experimental Design

The detection conditions for spectinomycin hydrochloride were determined by simulating the peak area variation using response surface methodology (Figure 2) and considering the instrumental parameter ranges: nebulizer temperature 85°C, evaporator temperature 80°C, detector temperature 50°C, and gas flow rate 0.2 SLM.

A. Effect of Gas Flow Rate and Evaporator Temperature on Peak Area

B. Effect of Gas Flow Rate and Nebulizer Temperature on Peak Area

C. Effect of Nebulizer Temperature and Evaporator Temperature on Peak Area

Fig 2 Variation in Peak Area

Injection Precision

The stock solution of spectinomycin hydrochloride was diluted to 0.35mg/mL, and six consecutive injections were performed under the specified chromatographic conditions with chromatograms recorded. The relative standard deviations (RSD%) of the retention time and peak area for the spectinomycin hydrochloride chromatographic peak were calculated. The results are showed in Table 5.

Table 5 Injection Precision Test Results

Specificity

According to the ICH Q2(R2) guideline on analytical procedure validation and the Chinese Pharmacopoeia General Chapter 〈9101〉 Guidelines on Analytical Method Validation, the optimized method was validated for specificity, linear range, accuracy, and repeatability.

Purified water and the sample solution (0.35 mg/mL) were analyzed in parallel with chromatograms recorded. The results (Figure 3) showed no chromatographic peak at the retention time of spectinomycin hydrochloride in the purified water chromatogram, while the peak for spectinomycin hydrochloride in the sample solution achieved baseline separation from adjacent peaks (R > 1.5).

Fig 3 Analytical Results of Spectinomycin Hydrochloride Sample (0.35mg/mL)

Linear Range

Prepare three parallel samples of spectinomycin hydrochloride stock solution according to the standard solution preparation method, analyze them under chromatographic conditions, and record the chromatograms. Perform regression analysis using the chromatographic peak areas and corresponding concentrations.

Fig 4 Linearity Test Results (Log-Transformed vs. Untransformed)

According to the literature, the relationship between the ELSD response value and concentration is nonlinear, requiring logarithmic transformation of both the response value and the concentration before performing regression analysis [8,9,10]. After applying logarithmic transformation to the experimental data, the correlation coefficient of the linear regression curve exceeded 0.999, which is significantly better than the result obtained without logarithmic transformation (as shown in Figure 4).

Accuracy and Repeatability

The spectinomycin hydrochloride stock solution was diluted to concentrations of 0.25, 0.35, and 0.50mg/mL. For each concentration, three parallel samples were prepared, analyzed under the specified chromatographic conditions, and the chromatograms were recorded. The content was calculated using the regression curve, and the recovery and repeatability were evaluated. The results are presented in Table 6.

Table 6 Recovery Test Results

Limit of Quantification (LOQ)

Dilute the streptomycin hydrochloride stock solution to a concentration of 12μg/mL. Analyze the sample under chromatographic conditions and record the chromatogram. Calculate the signal-to-noise ratio (S/N) of the streptomycin hydrochloride chromatographic peak and determine its repeatability. The results are shown in Table 7.

Table 7 LOQ Test Result

The results indicate that under the ICH guideline requirement of S/N > 10, the relative standard deviation of the peak area for this concentration of spectinomycin hydrochloride solution meets the criteria. Therefore, it can serve as the quantitative limit standard for spectinomycin hydrochloride.

5. Conclusion

Based on the pharmacopoeial method, this study established a reliable and robust analytical procedure for the quantification of spectinomycin hydrochloride. The evaporative light-scattering detector (ELSD) conditions were systematically optimized. The optimized method demonstrated improved stability and accuracy, higher detection sensitivity, and a satisfactory linear range.

The ELSD3260 detector is a newly designed and developed low-temperature, split-flow detector. It is suitable for the analysis of both non-volatile and semi-volatile substances, providing a valuable alternative for compounds lacking UV absorption. In this study, the use of the ELSD3260 for the analysis of spectinomycin hydrochloride resulted in excellent response values and reproducibility, along with enhanced detection sensitivity. These findings demonstrate that this instrument is well-suited for the quantitative analysis of spectinomycin hydrochloride.