This study mainly uses Shanghai Volmi Science and Technology Mobile Star I continuous flow analyzer to determine cyanide in liquor by continuous flow method. The national standard method "Determination of Cyanide in Food Safety National Standard" (GB 5009.36-2023) is used to verify the method. The determination objectives of this experiment include standard curve, blank sample, actual commercial liquor sample and standard addition recovery rate. The results show that: the standard curve, method detection limit and quantitative limit, precision and standard recovery and other indicators meet the requirements of the method, indicating that the reasonable use of continuous flow analyzer to determine the cyanide content in liquor, the operation difficulty is low and can meet the actual detection of diversity, trace, and get accurate results.

Keywords: continuous flow analyzer; cyanide; liquor; method validation

Abstract: In this study mainly used the Shanghai Fumi Technology Flow Star I continuous flow analyzer to measure cyanide in Baijiu and conducted method verification on the national standard method "National Food Safety Standard Determination of Cyanide in Foods" (GB 5009.36-2023).

Abstract: In this study mainly used the Shanghai Fumi Technology Flow Star I continuous flow analyzer to measure cyanide in Baijiu and conducted method verification on the national standard method "National Food Safety Standard Determination of Cyanide in Foods" (GB 5009.36-2023). and the standard curve，standard substance，blank sample，actual sample and standard addition were measured. The results show that: standard curve，detection limit，accuracy，precision and other indicators meet the requirements of the method. It shows that when the continuous flow analyzer is used rationally to measure the cyanide content in Baijiu, the operation difficulty is low, and it can meet the diversity and traceness of actual detection and obtain accurate results.

Key words: continuous flow analysis; cyanide; Baijiu; verification of the method

1 Introduction

Cyanide (Cyanide) is specifically refers to containing cyano (-CN) or cyanide ion (CN-) belongs to the highly toxic class of compounds, acute cyanide poisoning cyanide will be affected by the role of gastric acid, hydrolysis of hydrogen cyanide and absorbed into the blood. Ferrovalent iron (Fe3) in cytochrome c oxidase binds to cyanide in the blood to form cyanide-iron cytochrome oxidase, which causes Fe3 to lose the function of transporting electrons, which in turn leads to the interruption of the respiratory chain and prevents cells from breathing and death [1]. Since the main raw materials in liquor are made from cassava and wild plants, it will be hydrolyzed during fermentation to produce hydrocyanic acid. Therefore, the cyanide content of this liquor is relatively high. However, the amount of cyanide in wine made from grain raw materials is very small. Although most of the produced hydrocyanic acid can be volatilized by exhaust gas during the cooking process of raw materials, a small part of hydrocyanic acid remains in liquor [2]. In addition, the use of pesticides in part contaminated raw materials, as well as the process of winemaking mutation reactions are possible causes of cyanide contamination [3].

The National Food Safety Standard Distilled Liquor and Its Liquor (GB 2757-2012)[4] stipulates that the cyanide limit should be less than 8.0 mg/L (calculated by HCN and converted by 100 vol alcohol). At present, the main detection methods of cyanide in liquor are spectrophotometry, titration analysis, electrochemical detection, gas chromatography and continuous flow analysis. Compared with other detection methods, such as long time-consuming, large error and unstable detection results, continuous flow analysis method realizes the automatic detection of cyanide, which can avoid the unstable factors of manual operation, and can also save a lot of time, improve accuracy, and reduce the impact on the liquor matrix. Therefore, according to the latest detection standard of cyanide in liquor "national food safety standard determination of cyanide in food" (GB 5009.36-2023)[5], the method is verified to prove its accuracy, precision and so on to meet the detection of cyanide in liquor, so as to facilitate the analysis and detection of large quantities of samples.

2 Principle

Principle of 2.1 method

Since the alkaline hydrolysis of cyanide in liquor samples occurs in an acidic environment (PH = 3.8), cyanide can be hydrolyzed into hydrocyanic acid by on-line distillation at 125 ℃. At this time, gaseous hydrocyanic acid will be absorbed by sodium hydroxide solution. Then, in a weakly acidic environment, cyanide ions react with ammonium chloride T to generate cyanogen chloride, then react with isonicotinic acid (Isonicotinic acid), and finally undergo hydrolysis to generate pentenedial (Pentenedialdehyde acid), pentenedial and two 1,3-dimethylbarbituric acid (1,3-Dimethylbarbituric acid) molecular condensation reaction generates a red complex (the reaction mechanism is shown in fig. 1), which has a maximum absorption at a wavelength of 600 nm, and then enters the colorimetric cell for quantitative determination (see fig. 2 for the method analysis workflow of cyanide in liquor), which is compared with the standard series for quantitative determination. The linear range of the method was 0.002~1000 mg/L CN-, and the detection limit was 0.015 mg/L CN-. For the samples whose concentration was higher than the upper limit of the above determination, the samples could be diluted and then determined, and the analysis frequency of 22 samples was 1 h.

Fig.1 Determination of cyanide reaction texture in liquor samples by isonicotinic acid-barbituric acid spectrophotometry

2 Flow chart of continuous flow-isonicotinic acid-barbituric acid spectrophotometry for determination of cyanide in liquor samples

Principle of 2.2 instrument

The flow star I continuous flow analyzer produced by Shanghai vumi technology co., ltd. uses parallel flow technology to squeeze the elastic polyethylene (PE) pump tube through a peristaltic pump, and inject a certain proportion of sample samples, reagents and air into the closed pipeline reaction system, thus ensuring uniform and stable interval distribution of air bubbles, and enabling effective reproduction and accuracy of experimental results. Air bubble segmentation technology allows each segment to remain intact, reducing diffusion and sample passing, while cleaning the surface in the pipeline. In addition, the distribution of bubbles can also accurately reveal the source of all system problems [7].

In the process of pipeline flow, the sample and reagent can be mixed smoothly and produce chemical action. To achieve uniform mixing and chemical reactions, in the chemical analysis module, basically one or more online processing devices will be configured, so as to more conveniently realize various treatments such as online distillation, extraction and heating, and then generate the substances to be detected, flow into the detection module (colorimetric cell) for colorimetric detection, the instrument records different signal changes in each period, and the software automatically calculates the concentration value. Compared with manual operation, online sample processing makes the efficiency of the analysis process has been effectively improved, but also makes the reliability of the analysis has been strengthened, which helps to save manpower and material resources, reduce the consumption of samples and reagents, and the output of experimental waste liquid, which can provide fast and accurate analysis of a large number of samples for a large number of samples.

In the process of measuring a sample, the flow star I continuous flow analyzer can choose to measure one parameter by a single chemical reaction module, or two or more chemical reaction modules in parallel, and carry out simultaneous measurement on these modules, so as to achieve higher analysis efficiency and improve the test speed.

3 Experimental part

3.1 instruments and reagents

3.1.1 Main reagents, materials and instruments

The reagents used in the experiment are shown in Table 1, and the details of the experimental device are shown in Table 2.

Table 1 Reagents and materials

Table 2 Details of experimental device

3.1.2 Reagent preparation

The reagents used in this method are analytically pure and above, and the water used is ultra-pure water treated by Aquaplore 2S ultra-pure water machine.

3.1.2.1 isonicotinic acid/barbituric acid chromogenic agent (R1)

Weigh 4.4g of sodium hydroxide and dissolve it in 400mL of water. When it is completely dissolved, weigh 6.8g of 1,3-dimethylbarbituric acid and add it. After ultrasonic dissolution is complete, weigh 6.8g of isonicotinic acid and stir to dissolve. The pH value was adjusted to 5.5 with 0.5mol/L sodium hydroxide and diluted to 500mL. After vigorous stirring (e. g., using a magnetic stirrer) for 1 hour at 30°C and then filtration, it is stable for three months at 5°C.

3.1.2.2 Chloramine-T solution (R2)

Weigh 1g of chloramine-T, dissolve in 400mL of ultrapure water, dilute to 500mL, and place in a brown glass bottle until the solution is mixed and stable for one week.

3.1.2.3 Buffer solution (R3)

Weigh 13.6g of potassium dihydrogen phosphate and 0.34g of potassium dihydrogen phosphate and ultrasonically dissolve in 800mL of ultrapure water, adjust the pH value to 5.2 with 0.5mol/L sodium hydroxide, dilute to 1000mL with ultrapure water, finally add 3.0 mL 30% Brij35 solution, mix well, place in a brown glass bottle and stabilize at 4 ℃ for three months.

3.1.2.4 Absorption reagent (0.1N sodium hydroxide)(R4)

Weigh 4g sodium hydroxide and ultrasonically dissolve it in 500mL ultrapure water, and dilute it to 1000mL.

3.1.2.5 Distillation Reagent (R5)

Add 250mL of phosphoric acid into 500mL of ultrapure water, add 50mL of hypophosphorous acid, mix well, and dilute to 1000mL with ultrapure water.

3.2 sample pretreatment

According to the fifth method of "Determination of Cyanide in Food of National Food Safety Standard" (GB 5009.36-2023), the sample pretreatment method for distilled liquor, its configured liquor and edible alcohol is as follows: accurately suck 1.00 mL sample (including sesame-flavor liquor, Maotai-flavor liquor and special-flavor liquor sample 0.25 mL) into a 25 mL volumetric flask, use sodium hydroxide solution (1g/L) to fix the volume to the scale and mix evenly. After placing for 10 min, the sample is subjected to alkaline hydrolysis and then sample injection for determination. Since this experiment only uses three kinds of fragrance, such as rice fragrance type, fragrance type and Luzhou fragrance type, the sample pretreatment is carried out by the method of dilution multiple of 25.

3.3 experiment

3.3.1 Preparation of standard curve

Take 10.00mL of cyanide standard solution in water into a 100mL volumetric flask, and use 0.01N NaOH solution to fix the volume to the marking line to obtain a standard intermediate solution with a concentration of 5.00mg/L; Transfer 20.00mL of standard intermediate solution, and similarly use 0.01N NaOH solution to fix the volume into a 100mL volumetric flask to obtain a standard liquid with a concentration of 1.00mg/L; Take a group of 100mL volumetric flask, transfer 0.00mL, 0.10mL, 0.50mL, 1.00mL, 2.00mL, 5.00mL and 10.00mL of standard liquid, add them into volumetric flasks respectively, and fix the volume to the marking line with 0.01N NaOH solution, with concentrations of 0.00 mg/L, 0.001 mg/L, 0.005 mg/L, 0.010 mg/L, 0.020 mg/L, 0.050 mg/L and 0.100 mg/L respectively for later use.

3.3.2 Instrument conditions

The continuous flow analyzer is composed of an automatic sampler, a high-precision peristaltic pump, a chemical reaction unit, a colorimeter detection unit, a data processing unit, etc., and the instrument conditions for the determination of total cyanide are shown in Table 3 below.

Table 3 Instrument conditions for determination of cyanide in liquor by continuous flow analyzer of flow star I

3.3.3 Determination steps

(1) After connecting the air pipe, waste liquid pipe, cleaning pipe, etc. of the flow star I continuous flow analyzer to the designated position, connect the flow star I and the data line between the injector and the computer.

(2) Turn on the switch of the injector and the mobile star I detector, start the computer, check the "port" in the "resource manager" to confirm that the connection has been successful, and start the software to select the corresponding port.

(3) Start the cooling circulation tank, turn on the power switch first, then turn on the heating switch of the refrigeration and distiller to start heating; Check whether the pipeline connection is correct, cover the pump cover, start the pump and suck the reagent.

(4) Click the "Add" button to add the number of samples, click the "Run" button after the setting is completed, and finally enter the file name to save the new project.

(5) After the operation, close the distiller and heater to cool down the temperature, then take out all reagent pipelines from the reagent bottle, wipe the pipeline wall dry or rinse with ultra-pure water, and then put it into ultra-pure water for cleaning for 10min.

(6) After cleaning, close the peristaltic pump and open the pump cover to release the pressure of the pump pipe, close the switches of the flow star I, the sampler and the cooling circulation tank, and the experiment is over.

4 Results and discussion

4.1 standard curve and method detection limit

4.1.1 Standard curve

The concentration of cyanide standard solution in water is taken as the horizontal coordinate and the peak height is taken as the vertical coordinate, and the standard curve and correlation coefficient are calculated by software, as shown in Table 4. The experimental results show that according to the national food safety standard determination of cyanide in food (GB 5009.36-2023) by isonicotinic acid-barbituric acid continuous flow method for the determination of cyanide in liquor, the mass concentration in 0~0.100mg/L(50mm colorimetric cell) range showed a good linear relationship, the correlation coefficient r = 0.9999.

Table 4 Standard curve equation and correlation coefficient r for the determination of cyanide in liquor

4.1.2 Method detection limit

The detection limit experiment of this method is based on the determination method of method characteristic index in Appendix A of "Technical Guidelines for Formulating Standards of Environmental Monitoring and Analysis Methods" (HJ168-2020) [6]. According to all steps of sample analysis in the standard, the samples whose concentration or content is 3~5 times of the detection limit of the estimated method are determined n(n≥ 7) times in parallel, and the standard deviation of n times of parallel determination is calculated, calculate the detection limit of the method according to formula (1).

MDL = T (n-1,0.99) × S (1)

In the formula: MDL-method detection limit;

n-parallel determination times of sample;

t-the value of the t-distribution when the degree of freedom is n-1 and the confidence level is 99% (one-sided);

Standard deviation of S-n parallel determinations.

According to the standard, the standard with a concentration of 0.001 mg/L for 8 times was selected for this experiment. When n = 8, t(n-1,0.99)= 2.998[6]. The detection limit determination results are shown in Table 5. The calculation standard deviation is 0.039251, and the minimum detection limit is 0 .12 μg/L. According to the standard method verified, the quantitative limit is 4 times the method detection limit, and the quantitative limit is 0.47 μg/L. The limit of detection was 0.015mg/L(15 μg/L) and the limit of quantification was 0.050mg/L(50 μg/L), which was lower than the national food safety standard for determination of cyanide in foods (GB 5009.36-2023).

Table 5 Determination results of detection limit of total cyanide

4.2 method precision test

This experiment is divided into three concentration gradients of low concentration 0.005mg/L(1 μg/L), medium concentration 0.020mg/L(20 μg/L) and high concentration 0.100mg/L(100 μg/L) prepared by using certified standard substances. Among them, the purpose of low concentration is to determine the concentration near the lower limit, the medium concentration is the concentration near the middle point of the calibration curve, while the high concentration is the concentration near the upper limit of the linear range in the calibration curve. In addition, 5 kinds of wine samples are taken for detection, and 6 repetitive experiments are carried out for each concentration and wine sample according to the analysis steps and instrument conditions of the method. The liquor sample here is based on the "Determination of Cyanide in Food Safety National Standard" (GB 5009.36-2023). The content of cyanide (calculated by CN-) in the sample needs to be amplified by 25 times. The following table shows the original measured value without amplification.

Table 6 Precision of determination methods of liquor samples (n = 6)

The measurement results are shown in Table 6. The RSD(%) results of low, medium and high concentrations are in the range of 0.36-3.10%, and the RSD(%) results of 5 groups of commercial liquor samples with different concentrations are in the range of 0.36-3.82%, which all meet the requirements of the method.

4.3 standard recovery test

According to the method conditions of "Determination of Cyanide in Food Safety National Standard" (GB 5009.36-2023), dilution detection and analysis are carried out on different types of liquor sold on the market, in which S1 is rice flavor (alcohol concentration is 28% vol) and S2 is fen flavor (alcohol concentration is 35% vol), s3, S4 and S5 are of Luzhou-flavor type (alcohol concentration is 42% vol, 52% vol and 70% vol respectively). A small amount of background value is detected in different wine samples, and the content of cyanide (calculated as CN-) in the samples is 25 times larger than the original measured value. The standard addition recovery test was carried out on different types of liquors and concentrations of the detected substances. The standard addition was determined 6 times in parallel with the standard addition amounts of 0.005mg/L and 0.020mg/L. The results showed that the standard addition recovery rate was in the range of 86.3~109.4. In addition, the calculated relative standard deviation (RSD) was in the range of 0.44~2.45, indicating that the method was reliable and accurate. The results are shown in Table 7.

Table 7 Determination of liquor sample recovery and precision

5 Summary

5.1 standard curve evaluation

The correlation coefficient of the standard curve drawn in this experiment is r = 0.9999, which meets the requirements of the verification method "Determination of Cyanide in Food Safety National Standard" (GB 5009.36-2023)r≥ 999.

5.2 Detection Limit and Quantitative Limit Evaluation

In this experiment, the detection limit of the method is 0.12 μg/L and the quantitative limit is 0.47 μg/L. The detection limit is 0.015mg/L(15 μg/L) and the quantitative limit is 0.050mg/L(50 μg/L).

5.3 method precision test evaluation

According to the results, the RSD(%) results of low, medium and high concentrations were in the range of 0.36-3.10%, and the RSD(%) results of 5 groups of commercial liquor samples with different concentrations were in the range of 0.36-3.82%, which met the requirements of the method.

Evaluation of 5.4 Recovery Test

According to the determination results, when the spiked amount is 0.005mg/L and 0.020mg/L, the spiked recoveries of different types of spirits and concentrations are 86.3%~ 97.4 and 88.8%~ 109.4%, respectively, and the determination results meet the requirements of the verified method.

5.5 Conclusion

In this experiment, the flow star I continuous flow analyzer produced by Shanghai Volmi Technology Co., Ltd. is used to verify the national standard method "Determination of Cyanide in Food Safety National Standard" (GB 5009.36-2023). All the test parameters, such as standard curve, method detection limit, accuracy and precision, have reached the requirements of the method, from the results, the automatic sampling and automatic analysis process of the flow star I continuous flow analyzer can effectively avoid the human error and safety in the traditional method experiment. The determination process is simple and the dosage of reagent and drug is small. It can be used to determine large quantities of samples by this method, which can better meet the diversity and trace of actual detection and obtain accurate results.

References:

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