Solid Sampling Pyrolysis Adsorption-Desorption Thermal Conductivity Method for Rapid and Simultaneous Detection of N and S in Seafood

In this work, a rapid method for the simultaneous determination of N and S in seafood was established based on a solid sampling absorption-desorption system coupled with a thermal conductivity detector. This setup mainly includes a solid sampling system, a gas line unit for controlling high-purity oxygen and helium, a combustion and reduction furnace, a purification column system for moisture, halogen, SO2, and CO2, and a thermal conductivity detector. After two stages of purging with 20 s of He sweeping (250 mL/min), N2 residue in the sample-containing chamber can be reduced to <0.01% to improve the device’s analytical sensitivity and precision. Herein, 100 s of heating at 900 °C was chosen as the optimized decomposition condition. After the generated SO2, H2O, and CO2 were absorbed by the adsorption column in turn, the purification process executed the vaporization of the N-containing analyte, and then N2 was detected by the thermal conductivity cell for the quantification of N. Subsequently, the adsorbed SO2 was released after heating the SO2 adsorption column and then transported to the thermal conductivity cell for the detection and quantification of S. After the instrumental optimization, the linear range was 2.0–100 mg and the correlation coefficient (R) was more than 0.999. The limit of detection (LOD) for N was 0.66 μg and the R was less than 4.0%, while the recovery rate ranged from 95.33 to 102.8%. At the same time, the LOD for S was 2.29 μg and the R was less than 6.0%, while the recovery rate ranged from 92.26 to 105.5%. The method was validated using certified reference materials (CRMs) and the measured N and S concentrations agreed with the certified values. The method indicated good accuracy and precision for the simultaneous detection of N and S in seafood samples. The total time of analysis was less than 6 min without the sample preparation process, fulfilling the fast detection of N and S in seafood. The establishment of this method filled the blank space in the area of the simultaneous and rapid determination of N and S in aquatic product solids. Thus, it provided technical support effectively to the requirements of risk assessment and detection in cases where supervision inspection was time-dependent.


Introduction
Nitrogen (Nitrogen, N) in aquatic products is an important source of high-quality proteins for humans [1][2][3][4], and the current detection method corresponding to the food protein content in China is the GB 5009.5-2016 National standard for food safety determination of protein in food [5]. This method needs to be decomposed by adding concentrated sulfuric acid at 420 • C under the condition of catalytic heating, and the ammonia produced combines with sulfuric acid to form ammonium sulfate. The ammonia is separated by alkalization distillation, and titrated with a sulfuric acid or hydrochloric acid standard titration solution after absorption with boric acid. The nitrogen content is calculated according to the consumption of acid, and then multiplied by the conversion factor to obtain the content of protein. The international detection methods of N content include the thermal conductivity method, the Kjeldahl Method, the Dumas combustion method, the Sei-micro Kjeldahl Method [6][7][8][9][10][11], and so on. The Kjeldahl method, with its simple operation and relatively low equipment cost, is still recognized by almost all countries and organizations today [6]. However, lots of chemical reagents, such as concentrated sulfuric acid, sodium hydroxide solution, and boric acid solution are required, which not only increases the cost of testing, but also contaminates the environment seriously and can cause potential harm to the health of humans [12][13][14]. The Dumas combustion method has the advantages of small sample consumption, with no sample digestion process and no reagent pollution, all while being faster and more efficient [14,15]; however, the popularity of the Dumas combustion method is far smaller than that of the Kjeldahl method for the moment, due to the high cost of the instruments [15,16].
In order to ensure the health of consumers, China has established specific regulations for food additives, including sulfur dioxide. It limits the scope of the SO 2 used in food and the maximum allowable residue dose. The desire to make food brighter leads to the an exceedance of sulfur dioxide in food products, which affects the health of consumers [17][18][19]. The current detection method corresponding to SO 2 in China is the GB 5009.  National standard for food safety determination of sulfur dioxide in food [20]. This standard is only applicable to dry food. In this method, the sample is acidified and distilled in a closed container, and the distillate is absorbed with a lead acetate solution. The absorbed solution is acidified with hydrochloric acid, and then titrated by the iodine standard solution. The sulfur dioxide content in the sample is calculated according to the amount of iodine standard solution consumed. The Infrared Spectroscopy method, high-temperature combustion method, and acid-base titration method, Eschka Method, and Coulomb Titration Method were often used to detect the amount of S content [21][22][23][24][25]. When S was detected by the infrared method, different S infrared pools were needed for high S content and low S content. The high-temperature combustion method, acid-base titration method, and Eschka Method have the advantages of high accuracy and good repeatability, with disadvantages such as time consumption and cumbersome operation. The Infrared Spectroscopy method and Coulomb Titration Method are used widely with the advantages of high accuracy, convenient operation, high automaticity, and short time [26].
The above detection methods were for the determination of either N or S, including different detectors and different instruments. Meanwhile, most detection methods required cumbersome pretreatment steps such as digestion or distillation and titration, resulting in many problems such as long detection time and environmental pollution during digestion or distillation. To the best of our knowledge, there is no standard method or relevant instrument for an adsorption-desorption thermal conductivity method for the simultaneous detection of N and S in seafood in China thus far. There are similar instruments abroad, but there is no method standard. The United States, Australia, Germany, Britain, and other countries have conducted in-depth research on coal elemental analysis methods [27][28][29][30][31][32]. Therefore, the research and establishment of a rapid detection method for solid injection are in full swing [33][34][35][36][37][38][39][40][41][42].
The solid sampling analytical method of N and S in seafood was established based on the principle of adsorption-desorption thermal conductivity analysis, in which only one thermal conductivity detector was employed for the instrumental integration. In addition, this setup is mainly composed of a solid sampling system, a gas line unit for controlling high-purity oxygen and helium, a combustion and reduction furnace, and a purification column system. After optimization, the limits of detection (LODs) for N and S were 0.66 µg and 2.29 µg, equivalent to 6.6 µg/g and 22.9 µg/g at a 100 mg sample size, respectively.
The total time of analysis was less than 6 min without the sample preparation process, fulfilling the fast detection of N and S in seafood.

Design of Two-Stage Purging
In the previous studies, one-stage purging always resulted in excessive nitrogen residue in the sample falling hole, thereby impacting the following measurement of S. So, we designed a two-stage purging to fulfill the simultaneous detection of N and S. Herein, the first stage of purging occurs when the sample enters the sample falling hole of the ball cup; then, the ball cup was rotated to make the sample falling hole face the inlet pipe, isolating it from the outside air. The first stage of purging can reduce the N 2 residue in the container to 1% at the highest. Subsequently, the second stage of purging was carried out to remove the residual N. After two stages of purging, N 2 residue in the sample-containing chamber can be reduced to <0.01% so as to avoid excessive nitrogen residue. As with the previous studies, helium, a kind of inert gas, was chosen as the purging gas with an appropriate flow rate to ensure that the wrapped sample would not be deformed or damaged. As a result, compared to the previous one-stage purging methods, the two-stage purge design gives the lowest N signal as shown in Figure 1, while effectively eliminating the air introduction into the sample falling hole.
trolling high-purity oxygen and helium, a combustion and reduction furnace, and a purification column system. After optimization, the limits of detection (LODs) for N and S were 0.66 μg and 2.29 μg, equivalent to 6.6 μg/g and 22.9 μg/g at a 100 mg sample size, respectively. The total time of analysis was less than 6 min without the sample preparation process, fulfilling the fast detection of N and S in seafood.

Design of Two-Stage Purging
In the previous studies, one-stage purging always resulted in excessive nitrogen residue in the sample falling hole, thereby impacting the following measurement of S. So, we designed a two-stage purging to fulfill the simultaneous detection of N and S. Herein, the first stage of purging occurs when the sample enters the sample falling hole of the ball cup; then, the ball cup was rotated to make the sample falling hole face the inlet pipe, isolating it from the outside air. The first stage of purging can reduce the N2 residue in the container to 1% at the highest. Subsequently, the second stage of purging was carried out to remove the residual N. After two stages of purging, N2 residue in the sample-containing chamber can be reduced to <0.01% so as to avoid excessive nitrogen residue. As with the previous studies, helium, a kind of inert gas, was chosen as the purging gas with an appropriate flow rate to ensure that the wrapped sample would not be deformed or damaged. As a result, compared to the previous one-stage purging methods, the two-stage purge design gives the lowest N signal as shown in Figure 1, while effectively eliminating the air introduction into the sample falling hole.

High-Temperature Decomposition
Real seafood samples must be ashed and decomposed to release analytes containing N and S for subsequent measurement. To investigate the effect of decomposition, a nori CRM (GBW10023) with N content (5.0 ± 0.3%) and S content (2.26 ± 0.14%) was employed under different decomposition temperatures and times, and the results are shown in Figure 2. The measured contents of N and S go up with the increase in decomposition temperature from 600 to 800 • C; then, a plateau appears from 800 to 1000 • C, indicating a complete release of analytes containing N and S from the sample matrix. The curves of the decomposition time for N and S both demonstrate the highest signals at 100 s heating at 900 • C, thereby revealing a sufficient sample decomposition, measured by observing the sample residue after heating. Considering the favorable analysis precision (<5.0%), 100 s heating at 900 • C was chosen as the optimized decomposition condition. Real seafood samples must be ashed and decomposed to release analytes containing N and S for subsequent measurement. To investigate the effect of decomposition, a nori CRM (GBW10023) with N content (5.0 ± 0.3%) and S content (2.26 ± 0.14%) was employed under different decomposition temperatures and times, and the results are shown in Figure 2. The measured contents of N and S go up with the increase in decomposition temperature from 600 to 800 °C; then, a plateau appears from 800 to 1000 °C, indicating a complete release of analytes containing N and S from the sample matrix. The curves of the decomposition time for N and S both demonstrate the highest signals at 100 s heating at 900 °C, thereby revealing a sufficient sample decomposition, measured by observing the sample residue after heating. Considering the favorable analysis precision (<5.0%), 100 s heating at 900 °C was chosen as the optimized decomposition condition.  Table 1. The carrier gas flow rate has an effect on the adsorption capacity of the adsorption column. The higher the flow rate, the lower the adsorption capacity. When the flow rate is too low, the sampling time becomes longer. The carrier gas pressure has an impact on the adsorption ability of the adsorption column. The greater the pressure, the stronger the adsorption ability. A high level of pressure results in high requirements for the furnace bearing force and pipeline sealing. If the pressure is too low, the components cannot be

Carrier Gas
The carrier gas flow rate has an effect on the adsorption capacity of the adsorption column. The higher the flow rate, the lower the adsorption capacity. When the flow rate is too low, the sampling time becomes longer. The carrier gas pressure has an impact on the adsorption ability of the adsorption column. The greater the pressure, the stronger the adsorption ability. A high level of pressure results in high requirements for the furnace bearing force and pipeline sealing. If the pressure is too low, the components cannot be completely adsorbed and the adsorption efficiency is reduced, which affects the results.
Based on the content of the tested sample nori CRM (GBW10023), the N content (5.0 ± 0.3%) and S content (2.26 ± 0.14%) were used to compare the response value changes between different Carrier gas flow rates (n = 6). The results are shown in Figure 3. A lower content, including 0.4% N and 2.2% S, was determined at a gas flow rate of 250 mL/min when compared to the standard value. A lower content, 21.6% and 51.3%, and 17.0% and 22.1% at gas flow rates of 200 mL/min and 300 mL/min for N and S, respectively, showed the worst accuracy. As such, a gas flow rate of 250 mL/min was chosen as the best.
22.1% at gas flow rates of 200 mL/min and 300 mL/min for N and S, respectively, sho the worst accuracy. As such, a gas flow rate of 250 mL/min was chosen as the best.  Table 1.

Establishment of Standard Curves
Standard substance nori CRM (GBW10023) was weighed at 2.24 mg, 4.25 mg mg, 10.81 mg, 16.51 mg, 21.78 mg, 25.40 mg, and 31.61 mg and then decomposed in t cups placed in a Ni-Cr chrome electric resistance furnace. The generated H2O and were adsorbed by two purification columns. NOx was reduced to N2 in a Ni-Cr ch electric resistance furnace and N2 was measured by a high-purity helium-loaded the conductivity detector.
The standard curve was generated by plotting the N and S concentrations versu fluorescent area of the electric resistance furnace. Calculated from the absolute co versus the signal intensities (peak area) of N and S, the equation obtained for N wa 0.0000643396x − 0.0187, R = 0.9991. This standard curve is shown in Figure S1. The e tion obtained for S was: y = 0.000197x − 0.0606, R = 0.9998, shown in Figure S2. Figu represents the standard curve of N and S.

Linearity Range, Recovery Rate, Precision, and Sensitivity of the Method
The linear range was 2.0-100 mg and the correlation coefficient (R) was more 0.999. The limit of detection (LOD) for N was 0.66 μg, equivalent to 6.6 μg/g at a 10 sample size (maximum sample size for sample boat), calculated from 3σ of 11 mea ments close to blank. The R was less than 4.0% and the recovery rate ranged from 95 to 102.8%. At the same time, the LOD for S was 2.29 μg, equivalent to 22.9 μg/g at mg sample size (maximum sample size for sample boat), calculated from 3σ of 11 m urements close to blank. The R was less than 6.0% and the recovery rate ranged 92.26% to 105.5%. The total time of analysis was less than 6 min.
The proposed method met the requirements of accurate and rapid N and S dete by the direct injection of aquatic product solids. The analytical performance of this in ment was in full accordance with the N and S content requirements of the assay, as sh in Tables 2 and 3. In Tables 4 and 5, the recovery rates of the N and S content of Pectin (GBW10024) (n = 12.8 ± 0.8%; s = 1.5 ± 0.1%) (n = 6) ranged from 100.2 to 102.8% and to 103.3% and RSD was less than 5.0%. For Fenneropenaeus chinensis (GBW10050) 13.5%; S = 1.0%) and ground fish powder, better assay accuracy was shown in Table  S4 and Figures S4-S6, respectively. The recovery rate for N in Fenneropenaeus chin (GBW10050) and ground fish powder ranged from 95.34 to 101.3% and 97.31 to 10 respectively. The recovery rate for S ranged from 92.26 to 100.2% and 96.93 to 10

Establishment of Standard Curves
Standard substance nori CRM (GBW10023) was weighed at 2.24 mg, 4.25 mg, 5.71 mg, 10.81 mg, 16.51 mg, 21.78 mg, 25.40 mg, and 31.61 mg and then decomposed in tinfoil cups placed in a Ni-Cr chrome electric resistance furnace. The generated H 2 O and CO 2 were adsorbed by two purification columns. NOx was reduced to N 2 in a Ni-Cr chrome electric resistance furnace and N 2 was measured by a high-purity helium-loaded thermal conductivity detector.
The standard curve was generated by plotting the N and S concentrations versus the fluorescent area of the electric resistance furnace. Calculated from the absolute content versus the signal intensities (peak area) of N and S, the equation obtained for N was: y = 0.0000643396x − 0.0187, R = 0.9991. This standard curve is shown in Figure S1. The equation obtained for S was: y = 0.000197x − 0.0606, R = 0.9998, shown in Figure S2. Figure S3 represents the standard curve of N and S.

Linearity Range, Recovery Rate, Precision, and Sensitivity of the Method
The linear range was 2.0-100 mg and the correlation coefficient (R) was more than 0.999. The limit of detection (LOD) for N was 0.66 µg, equivalent to 6.6 µg/g at a 100 mg sample size (maximum sample size for sample boat), calculated from 3σ of 11 measurements close to blank. The R was less than 4.0% and the recovery rate ranged from 95.33% to 102.8%. At the same time, the LOD for S was 2.29 µg, equivalent to 22.9 µg/g at a 100 mg sample size (maximum sample size for sample boat), calculated from 3σ of 11 measurements close to blank. The R was less than 6.0% and the recovery rate ranged from 92.26% to 105.5%. The total time of analysis was less than 6 min.
The proposed method met the requirements of accurate and rapid N and S detection by the direct injection of aquatic product solids. The analytical performance of this instrument was in full accordance with the N and S content requirements of the assay, as shown in Tables 2 and 3. In Tables 4 and 5, the recovery rates of the N and S content of Pectinidae (GBW10024) (n = 12.8 ± 0.8%; s = 1.5 ± 0.1%) (n = 6) ranged from 100.2 to 102.8% and 96.66 to 103.3% and RSD was less than 5.0%. For Fenneropenaeus chinensis (GBW10050) (N = 13.5%; S = 1.0%) and ground fish powder, better assay accuracy was shown in Tables S1-S4 and Figures S4-S6, respectively. The recovery rate for N in Fenneropenaeus chinensis (GBW10050) and ground fish powder ranged from 95.34 to 101.3% and 97.31 to 100.2%, respectively. The recovery rate for S ranged from 92.26 to 100.2% and 96.93 to 105.5%, respectively.

Detection and Validation of Real Sample
The Spirulina (GBW10025) (n = 10.6 ± 0.4%; s = 0.78 ± 0.08%) and shredded squid bought from malls were used to compare the detection results obtained in this study with the current method of testing N (GB 5009.5-2016) and S (GB 5009.  in food products in Table 6. The ANOVA test showed no significant difference (p > 0.05). In Table S5, more than 50 real samples including the types of dry, fresh, and aquatic products were detected to verify the methodological ability. The ANOVA test showed no significant difference (p > 0.05). It showed that this method was better than the national standard method due to the instruments and instrument condition parameters that we researched, and the selection was very reasonable and appropriate. Table 6. Comparison between this method and national standard methods for N and S content detection in food (n =6).

Instrumentation
This solid sampling nitrogen and sulfur analyzer (5e-chons2400) (Figure 4) mainly comprises a solid sampling system, a gas line unit for controlling high-purity oxygen and helium, a two-stage purge device, a combustion and reduction furnace, a purification column system for moisture, halogen, SO 2 , and CO 2 , and a thermal conductivity detector. The thermal conductivity cell section was equipped with a mass flow controller (MFC), gas path control system, and a reference gas flow controller and detector. The samples enter the high-temperature decomposition catalytic module for oxidative decomposition with oxygen, producing CO 2 , NO X , H 2 O, SO X , halide, and other gases. Then, in the reduction module, NO X and SO X react with agent copper particles to generate N 2 and SO 2 , respectively. Excess O 2 is absorbed through the reaction with copper particles to form CuO. The halogen compound is purified by the silver wire in the reduction tube. So far, the gas coming from the reduction tube only contains N 2 , CO 2 , H 2 O, and SO 2 . The mixed gas in the instrument is separated and purified successively by the adsorption-separation method (Figures 4 and 5). The mixed gas passes through SO 2 , H 2 O, and CO 2 adsorption devices to be absorbed in turn, and N 2 is left, and then N 2 is detected by the thermal conductivity cell for the quantification of N. Subsequently, SO 2 can be obtained by heating the SO 2 adsorption device after switching to the air path of SO 2 , and then SO 2 is detected by the thermal conductivity cell for the quantification of S.

Instrumentation
This solid sampling nitrogen and sulfur analyzer (5e-chons2400) (Figure 4) mainly comprises a solid sampling system, a gas line unit for controlling high-purity oxygen and helium, a two-stage purge device, a combustion and reduction furnace, a purification column system for moisture, halogen, SO2, and CO2, and a thermal conductivity detector. The thermal conductivity cell section was equipped with a mass flow controller (MFC), gas path control system, and a reference gas flow controller and detector. The samples enter the high-temperature decomposition catalytic module for oxidative decomposition with oxygen, producing CO2, NOX, H2O, SOX, halide, and other gases. Then, in the reduction module, NOX and SOX react with agent copper particles to generate N2 and SO2, respectively. Excess O2 is absorbed through the reaction with copper particles to form CuO. The halogen compound is purified by the silver wire in the reduction tube. So far, the gas coming from the reduction tube only contains N2, CO2, H2O, and SO2. The mixed gas in the instrument is separated and purified successively by the adsorption-separation method (Figures 4 and 5). The mixed gas passes through SO2, H2O, and CO2 adsorption devices to be absorbed in turn, and N2 is left, and then N2 is detected by the thermal conductivity cell for the quantification of N. Subsequently, SO2 can be obtained by heating the SO2 adsorption device after switching to the air path of SO2, and then SO2 is detected by the thermal conductivity cell for the quantification of S.

Analytical Procedures by Solid Sampling System
The analytical procedures of the solid sampling system are summarized in Table 1, including two-stage purging, high-temperature decomposition, adsorption-desorption, and detection. (1) Two-stage purging: the sample was weighed into the sampling boat and after 20 s of He sweeping (250 mL/min) to reduce nitrogen residue. (2) High-temperature decomposition: the sample was inserted into the high-temperature decomposition catalytic module at 900 • C pyrolysis for 100 s for oxidative decomposition with oxygen, producing CO 2 , NO X , H 2 O, SO X , halide, and other gases. (3) Adsorption-desorption: The mixed gas was transported into the bottom of the combustion tube with reduced copper to generate Molecules 2022, 27, 8909 8 of 11 N 2 and SO 2 and excess O 2 was absorbed. Subsequently, the mixed gas passes through SO 2 , H 2 O, and CO 2 adsorption devices to be absorbed in turn. SO 2 was adsorbed by the adsorption column fitted with carbon molecular sieves; H 2 O was captured by the first gas purification column filled with magnesium perchlorate and phosphorus pentoxide; CO 2 was captured by the second purification column packed with alkali asbestos and phosphorus pentoxide. Finally, N 2 was left to be detected. (4) N Detection: N 2 was detected within 6 s by the thermal conductivity cell for the quantification of N under the conditions of 8 KΩ thermal resistance, a 7× amplification circuit, 250 mL/min carrier gas, and 50 mL/min reference gas. (5) S Detection: After completing N Detection, the captured SO 2 was released once the SO 2 adsorption column was heated, and then it was transported to the thermal conductivity cell for the detection and quantification of S.

Analytical Procedures by Solid Sampling System
The analytical procedures of the solid sampling system are summarized in Table 1, including two-stage purging, high-temperature decomposition, adsorption-desorption, and detection. (1) Two-stage purging: the sample was weighed into the sampling boat and after 20 s of He sweeping (250 mL/min) to reduce nitrogen residue. (2) High-temperature

Sample Preparation
The edible sites from living fresh aquatic products were made into homogenate using a T18 homogenizer (IKA, Königswinter, Germany). The dry aquatic product was pulverized to a powder for use. The fresh sample, with a weight of about 50.00 mg, was weighed using a tinfoil cup using a Cp2250 electronic analytical balance (sartorius, Goettingen, Germany), and the powder sample weighed about 10 mg (depending on its N and S content). Then, their N and S content was determined in the machine.

Conclusions
In this work, a novel solid sampling method for the rapid and simultaneous detection of N and S in seafood was for the first time developed. The two-stage purging method was newly designed to avoid excessive nitrogen residue, so as to improve analytical sensitivity and precision. Thus, the method LOD for N and S reached 0.66 µg and 2.29 µg, respectively. The recovery rate of N and S ranged from 95.33 to 102.8% and 92.26 to 105.5%, respectively. The whole analysis time was <6 min without the sample preparation process. Herein, only one thermal conductivity detector was employed for further instrumental integration. This method has promising application potential in the sensitive, rapid, green, and robust detection of N and S to evaluate the nutritional quality of seafood.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27248909/s1, Figure S1: Standard curve of N ranging from 2.24 mg to 31.61 mg; Figure S2: Standard curve of S ranging from 2.24mg to 31.61mg; Figure S3: Spectrum of N and S in a nori CRM sample (GBW10023); Figure S4: Spectrum of N and S in Standard substance Pectinidae (GBW10024); Figure S5: Spectrum of N and S of Fenneropenaeus chinensis (GBW10050); Figure S6: Spectrum of N and S in grilled fish flakes; Table S1: Results of N recovery rate in live Fenneropenaeus chinensis flesh; Table S2: Results of S recovery rate in live Fenneropenaeus chinensis flesh; Table S3: Results of recovery rate of N content in ground fish powder; Table S4: Results of recovery rate of S content in ground fish powder; Table S5: Comparison between this method and national standard methods for N and S content detection in many seafood species (n = 30).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the study population to publish this paper.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the privacy of the study participants.