Determination of Resin Residues in Panax Notoginseng Saponins by Wayeal Gas Chromatograph GC6100

Gas chromatography plays a critically important role in the detection of resin residues in Panax notoginseng saponins. Panax notoginseng saponins serve as the core raw material for cardiovascular and cerebrovascular drugs such as Xueshuantong, and their purification process commonly employs macroporous adsorption resins. However, this process may introduce harmful organic solvent residues, including n-hexane, benzene, toluene, xylene, styrene, and divinylbenzene. These residues are potentially toxic to the human body; therefore, it is essential to establish sensitive and accurate analytical methods for stringent monitoring.

This study was conducted in accordance with the latest edition of the Chinese Pharmacopoeia, utilizing the Wayeal Technology GC6100 gas chromatograph equipped with a flame ionization detector (FID) and coupled with an automated headspace sampler for the determination of resin residues in Panax notoginseng saponin samples.

Keywords: Panax notoginseng saponins; residual solvents; gas chromatography; FID detector; headspace.

1. Experiment Method

1.1 Instrument Configuration

Table 1 Configuration List of Gas Chromatograph

1.2 Experimental Materials and Auxiliary Equipment

Reference standards: Certified reference materials of n-hexane, benzene, toluene, p-xylene, o-xylene, styrene, 1,2-diethylbenzene, and divinylbenzene standard solutions (purchased in-house), stored in sealed containers under refrigeration and protected from light;

N,N-dimethylformamide;

Carrier gas: High-purity nitrogen;

Hydrogen generator;

Air generator;

Automated headspace sampler;

Headspace vials: Glass headspace vials (20 mL).

1.3 Test Conditions

1.3.1 Reference Conditions for the Headspace Sampler

Oven temperature: 90°C;

Incubation time: 30 min;

Injection valve temperature: 110°C;

Transfer line temperature: 120°C;

Injection volume: 1.0mL (loop volume).

1.3.2 Reference Operating Conditions for the Gas Chromatograph

Analytical column: FFAP capillary column, 30m × 0.25mm × 0.25μm

Temperature program: Initial column temperature of 60°C held for 16 min, then ramped to 200°C at a rate of 20°C/min, and held for 2 min;

Column flow rate: 1.0 mL/min;

Inlet temperature: 240°C;

Detector temperature: 300°C;

Air flow rate: 300mL/min;

Hydrogen flow rate: 40mL/min;

Makeup flow rate: 20mL/min;

Split injection with a split ratio of 2:1.

1.4 Solution Preparation

1.4.1 Preparation of Reference Standard Stock Solution

Accurately weigh appropriate amounts of the reference standards of n-hexane, benzene, toluene, p-xylene, o-xylene, styrene, 1,2-diethylbenzene, and divinylbenzene, and dissolve in N,N-dimethylformamide to prepare a solution containing 20 μg/mL each of n-hexane, toluene, p-xylene, o-xylene, styrene, 1,2-diethylbenzene, and divinylbenzene, and 4 μg/mL of benzene, respectively, serving as the reference standard stock solution.

1.4.2 Preparation of Mixed Standard Working Solution

Accurately transfer 2mL of the reference standard stock solution into a 50mL volumetric flask, dilute to volume with 25% N,N-dimethylformamide solution, and mix well. Accurately transfer 5mL of this solution into a 20mL headspace vial, seal, and the mixed standard working solution is obtained.

1.4.3 Preparation of Standard Solution for Detection Limit Test

Accurately transfer 2mL of the reference standard stock solution into a 500mL volumetric flask, dilute to volume with 25% N,N-dimethylformamide solution, and mix well. Accurately transfer 5mL of this solution into a 20mL headspace vial, seal, and the standard solution for the detection limit test is obtained.

2. Result and Discussion

2.1 Qualitative Analysis of Reference Standards

Fig 1 Chromatogram of the Blank Solvent

Fig 2 Chromatogram of the Mixed Standard Working Solution

Table 2 Chromatographic Parameters of the Mixed Standard Working Solution

Note: The above chromatograms shown that all chromatographic peaks are well separated from each other, with resolutions greater than 1.5, meeting the requirements for experimental analysis.

2.2 Repeatability Test

Fig 3 Overlaid Chromatograms of the Mixed Standard Working Solution for Repeatability Test (n=5)

Table 3 Chromatographic Parameters of the Mixed Standard Working Solution for Repeatability Test

Note: The mixed standard working solution was continuously injected for 5 times, and the RSD of peak areas was ≤1.5%.

2.3 Detection Limit

Fig 4 Chromatogram of the Standard Solution for Detection Limit Test

Table 4 Chromatographic Parameters of the Standard Solution for Detection Limit Test

Note: The limit of detection (LOD) was determined at a signal-to-noise ratio (S/N) of 3, and the limit of quantitation (LOQ) was determined at a signal-to-noise ratio (S/N) of 10. Both the LOD and LOQ values were found to meet the requirements of the standard.

2.4 Sample Analysis

Accurately weigh approximately 0.1g of each sample (0.1017g, 0.1026g, and 0.1023g, respectively) into separate 20mL headspace vials, then accurately add 5mL of 25% N,N-dimethylformamide solution to each vial, seal, and shake well to mix.

Fig 5 Overlaid Chromatograms of the Three Sample Solutions

Fig 6 Overlaid Chromatograms of the Sample Solution and the Reference Standard Solution

Note: None of n-hexane, benzene, toluene, p-xylene, o-xylene, styrene, 1,2-diethylbenzene, or divinylbenzene were detected in the samples.

3. Conclusion

In this study, a Wayeal GC6100 gas chromatograph equipped with a flame ionization detector (FID) and coupled with an automated headspace sampler was employed for the determination of resin residues in Panax notoginseng saponin samples. The experimental results demonstrated that all chromatographic peaks were well separated, with resolutions greater than 1.5, meeting the requirements for the analytical purposes. The repeatability and detection limit test results of this method all met the specified requirements. None of n-hexane, benzene, toluene, p-xylene, o-xylene, styrene, 1,2-diethylbenzene, or divinylbenzene were detected in the test samples, indicating satisfactory results. These results demonstrate that the Wayeal GC6100 gas chromatograph is fully capable of meeting the analytical requirements for the determination of resin residues in Panax notoginseng saponin samples.

4. Attention

All operations involving the preparation of solvent and standard solutions, as well as sample pretreatment, should be carried out in a fume hood. Appropriate personal protective equipment should be worn in accordance with laboratory safety regulations to avoid contact with skin and clothing.