r vacuum distillation), multiple extractions of N-nitrosamines from the distillate with methylene chloride, preconcentration by evaporation, and direct analysis for chromatography-mass spectrometry and chemiluminescence method   . No scientific papers reported using the Reverse-Phase HPLC method and direct UV detection when extracting N-nitrosamines by autoclave treatment. However, the literature reveals a very few methods which have been published for quantification of N-nitrosamines in meat products using HPLC with direct UV detection and all the reported methods were adopting pre-column or post-column photolysis units or additional derivatization reaction to enhance the light absorbance or to get fluorescent derivatives    . First attempts to use HPLC for the determination of N-nitrosamines included reversed-phase HPLC with the UV detection of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD chloride), 8-methoxy-5-quinolinesulfonylaziridine (QAZ) or 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride) derivatives of secondary amines obtained on the denitrosation of N-nitrosamines  . Some authors proposed to use post-column photolysis and derivatization. N-nitrosamines are separated from the sample matrix using reversed-phase liquid chromatography. The N-nitroso bond is cleaved by UV photolysis with the formation of nitrite ion then the nitrite is diazotized with sulfanilamide in an acid medium (according to the Griess reaction), and is then coupled with N-(1-naphthyl) ethylenediamine dihydrochloride (NED) to form a purple-coloured azo dye that is quantitatively determined spectrophotometrically at a maximum wavelength, λmax, of 540 nm   .
The aim of this work was the development of a RP-HPLC with direct UV-detection method for the determination of traces of seven volatile n-nitrosamines simultaneously from meat samples after previous autoclaving treatment  . The developed method was validated according to International Conference on Harmonization (ICH) guidelines to show the capability of the method  .
EPA 521 nitrosamine mix standard was purchased from Supleco (USA), this solution contained seven analytes at 2000 µg/mL of each: N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitroso-diethylamine (NDEA), N-nitrosopyrrolidine (NPYR), N-nitrosodin-propylamine (NDPA), N-nitrosopiperidine (NPIP) and N-nitrosodi-n-butylamine (NDBA), and individual standards to each of the Nitrosamines with a concentration of 5000 µg/mL from Sigma Aldrich. For sample preparation, sodium hydroxide, ethanol, octane and dichloromethane (DCM) were purchased from Sigma-Aldrich (USA). All other chemicals (ascorbic acid, anhydrous sodium sulfate and sodium chloride) used in this research were of analytical laboratory grade acetonitrile, Methanol, and water (HPLC grade), ammonium hydroxide acid and HPLC water were obtained from Sigma Aldrich (USA). The 0.22 µm PVDF syringe filter was purchased from Millipore (India). Raw meat samples purchased from local supermarkets in Damascus.
Pyrex tubes (20 ml) with heat-stable Teflon-lined caps were used. Glass column (30 cm × 1.5 cm). Kuderna Danish (KD) concentrator was used for the concentration of organic solvents. Autoclave (Selecta, Spain, 4001757). CAMAG UV Lamp dual wavelength, 254/366 nm, 2 × 8 watt in combination (Switzerland). KNAUER® HPLC system (KNAUER®, Germany), consisting of smart line manager, sample manager, and smartline UV detector 2500. System control, data collection, and data processing were accomplished using EurochromTM chromatography data software.
2.3. Chromatographic Conditions
The chromatographic condition was optimized using the Knauer Symmetry C18, 5 µm (100 mm × 4.6 mm) column. A solvent A is 10 mM Ammonium hydroxide and acetonitrile as solvent B. This was then filtered through a 0.22 µm PVDF membrane filter and degassed under vacuum prior to use. The separation of all components was achieved by gradient elution using solvent A and B. Solution A was used as diluent. The final selected and optimized conditions were as follows; injection volume 20 µL, gradient elution (solvent B from 0 to 90% during 10 min then maintained to the end of the run), at a flow rate of 1.0 mL/min at 80˚C (column oven) temperature, detection wavelength 231 nm, and sample temperature 15˚C. Under these conditions, the backpressure in the system was about 2500 psi.
2.4. Sample Preparation
Extraction of N-nitrosamines was performed using the same method as our previous work  with a modification at the end. Approximately 6 grams of meat sample was placed in the Pyrex tube into which 10 mL of sodium hydroxide 1N was poured. The tube was capped tightly and autoclaved at 121˚C for 10 min. After being allowed to stand at room temperature, the autoclaved solution was transferred to 50 mL separatory funnel. The tubes was rinsed twice with 5 mL of ethanol and then 10 mL of dichloromethane, and the rinsing solutions and 10 mL of 10% aqueous sodium chloride were combined with the original extract in the separatory funnel. After being shaken, the dichloromethane layer was collected, and the water layer was re-extracted with 10 ml of dichloromethane. The dichloromethane extracts were combined, dried over anhydrous sodium sulfate and concentrated to approximately 0.5 mL using KD concentrator and nitrogen gas flow. The concentrate was loaded onto a silica gel column (30 cm × 1.5 cm) (equilibrated with dichloromethane) and the column was eluted with 10 ml of dichloromethane. After the addition of 100 µL of octane (to prevent exsiccation of the solvent), the elute was concentrated to 1 mL using KD concentrator and nitrogen gas then extracting 3 mL methanol. This was repeated three times. The combined methanol extracts were concentrated to about 100 µL under a nitrogen stream.
2.5. Preparation of N-Nitrosamine Mix Standard
A series of working standard solutions were prepared by appropriate dilution of the EPA 521 nitrosamine mix with solvent A and stored at –20˚C before use. From the primary stock solution 2000 µg/mL of each nitrosamine, a 1/50 dilution was done to get 40 µg/mL secondary stock solution of each nitrosamine. Sequentially dilute secondary stock solution was performed to get standards titrating at 0.5, 1, 2.5, 5, 7.5,10, and 15 µg/mL in 100 mL volumetric flasks, these solutions kept in the absence of light.
3. Results and Discussions
3.1. Method Development and Optimization
The main criteria for development of a RP-HPLC method for the determination of seven N-nitrosamine compounds in raw meat and the method should be able to quantify all seven compounds in a single run and should be accurate, precise, linear, free of interference from blank, and enough for the routine use in quality control laboratories. Preliminary experiments were carried out until separation was achieved on C18 using injection volume of 20 µL, gradient elution (solvent B from 0% to 90% during 10 min then maintained to the end of the run), at a flow rate of 1.0 mL/min at 80˚C (column oven) temperature, detection wavelength 231 nm, and sample temperature 15˚C, slight low response and some base line disturbance for nitrosamines was observed when analyzing the samples. Therefore, a slight modification on sample preparation was done by extracting the nitrosamines with methanol as mentioned in sample preparation. The peak shape and response of all nitrosamines were improved. Finally, satisfactory result was achieved in 10 minutes with flow rate of 1.0 mL/min of the mobile phase. In order to judge the suitability of method for determining the N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N nitrosopyrrolidine (NPYR), N-nitrosodinpropylamine (NDPA), N-nitrosopiperidine (NPIP) and N-nitrosodi-n-butylamine (NDBA) traces in meat, the method was validated as per the ICH guideline for specificity, limit of detection, limit of quantification, linearity, accuracy and precision  . The matrix effect was studied by comparing the slope of the aqueous standards and standard additions calibration graphs obtained for the meat samples, no statistically differences were observed, and so quantification was carried out by external calibration. Calibration curves were obtained by least squares linear regression analysis for the peak area versus the analyte concentration using six concentration levels in duplicate. The method was validated to demonstrate that it is suitable for its intended purpose by the standard procedure to evaluate adequate validation characteristics. Retention times of selected N-nitrosamines were determined by using standard solutions of each nitrosamine. Figure 1 shows the chromatogram obtained by HPLCUV of unspiked meat sample.
Specificity is the ability of the method to measure the analyte response in the presence of diluent. Figures 1 and 2 show that there is no interference at the RT (retention time) for all seven nitrosamine compounds due to the blank, and Table 1 shows the summery peak purity results. To assess the ability of the method, Mix standard solution were prepared with known amounts of NDMA, NMEA, NDEA, NPYR, NDPA, NPIP, NDBA, and injected into the HPLC and the chromatograms were recorded. The sample (raw meat) solution was prepared as per the methodology and injected into the chromatograph (control sample). The sample shows no peaks either due to any nitrosamines. So it reveals that the raw meat (control sample) is free from nitrosamines under investigation. Therefore the sample extract was spiked with known amount of each nitrosamine reference standard at target level (1 µg/ml), and injected into the chromatograph (Spiked sample). The relative retention time for NDMA, NMEA, NDEA, NPYR, NDPA, NPIP and NDBA is shown in Table 2. The resolution between the nearest compounds peak (NDPA and NDBA) was found to be 5.96 Therefore, no interference of blank was observed corresponding to any of nitrosamines. Therefore, the selectivity of the method was judged from the absence from the interfering peaks (false peaks) at the analyte elution times for blank chromatograms of different unspiked samples after irradiating them to UV light (wavelength 366 nanometer) for three hours to destroy nitrosamines if they are present  . The original signals were wrongly positive if the signals from the samples are not remarkably diminished after the irradiation. No matrix compounds existed that might give a false positive signal to the blank samples as shown in Figure 2.
Figure 1. Chromatogram of meat sample (unspiked and before irradiating).
Table 1. summery of peak purity results.
aRetention time; bNot applicable.
Table 2. Summary of system suitability parameter.
aN-nitrosodiethylamine; bN-nitrosomethylethylamine; cN-nitrosodiethylamine; dNnitrosodin-propylamine; eN-nitrosodi-n-butylamine; fN-nitrosopiperidine; gN-nitrosopyrrolidine; hnot applicable.
Figure 2. Chromatogram of blank (meat sample after irradiating).
System suitability parameters were measured to verify the system performance. System precision was determined by six replicate injections of standard preparation. Important characteristics including RSD %, resolution (between all nearest peaks), tailing factor, and theoretical plate number were measured. The RSD % of area counts of six replicate injections for all N-nitrosamine peaks were below 2.0%, and the resolution between the two nearest peaks was better than 1.5. These indicate that the system is precise and suitable for determination of all seven N-nitrosamine compounds. The results obtained are shown in Table 2. The parameters all complied with the acceptance criteria and system suitability was established.
The accuracy of an analytical method is the closeness of test results obtained by that method compared with the true values. To confirm the accuracy of the proposed method, recovery experiments were carried out by the standard addition technique. The accuracy of the method was carried out by adding known amounts of each N-nitrosamine corresponding to three concentration levels; 50%, 100%, and 150% of the target concentration to the sample examined (6 grams of the sample) in triplicate. The samples were given the same treatment as described in sample preparation procedure of validation. The percentage recoveries of all components at each level and each replicate were determined. The mean of percentage recoveries (n = 3) and the relative standard deviation were calculated. The amount recovered was within ±15.0% of the amount added, which indicates that the method is suitable for the determination of N-nitrosamines. It was confirmed from results that the method is accurate (Table 3).
The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of the analyte in a sample within a given range. The response function was determined by preparing standard solutions at six different concentration levels ranging from 1 - 15 µg/mL for all components (LOQ to 300% of the analyte concentration). The response was found to be linear from the (1 µg/mL) to (15 µg/mL) of the standard concentration. For all the compounds, the correlation coefficients were greater than 0.999. The linearity concentration and the regression statistics are shown in Tables 4 and 5 respectively. Figure 3 shows the chromatogram 1 µg/mL standard solution of each N-nitrosamine. The linearity curves for all components are presented in Figure 4.
Table 3. Accuracy results.
aRelative standard deviation.
Figure 3. Chromatogram of composite N-nitrosamines standard (1 µg/ml of each nitrosamine).
Figure 4. Linearity curves of tested N-nitrosamines.
3.6. Limit of Quantification (LOQ) and Limit of Detection (LOD)
The signal-to-noise ratio (S/N) method was adopted for the determination of the lower limit of quantification. The limit of quantification is estimated to be ten times the S/N ratio, limit of detection is estimated to be three times of S/N ratio. The quantification limit was achieved by injecting a series of possible dilute solutions of all components and the precision was established at the quantification level. The RSD % of peak areas was well within the acceptance limit, not more than 10%. The determined lower limit of qualification and precision at LOQ values for all components are presented in Table 6.
Table 6. LOQ and LOQ precision.
3.7. Sample Results
Meat samples were treated with 1 N NaOH for 10 min in autoclaved conditions and then analyzed by HPLC-UV and the results of 25 samples of different meat products which examined for the evaluation of the analytical method shows the presence of some volatile nitrosamine in all different products, and the absence of NMEA NDPA, and NDBA in 5 sample tested which may related to the presence of these three nitrosamines in low amounts which may below under the quantitation limit of our procedure, or the real absence of these nitrosamine in the sample tested. Statistically when applying (PASW Statistics 18) one way ANOVA test for statistical comparisons between the means of sum of seven nitrosamines in meat samples, there was significant difference (p < 0.05) which may related to the low number of samples, or because that some of tested samples are may be preserved by nitrite salts which contributing in nitrosamine formation in meat. The average sum of the N-nitrosamine found was in the range of 3.3 to 7 µg/kg of meat samples.
A gradient RP-HPLC method was successfully developed for the estimation of seven volatile N-nitrosamines in the meat products in single determination. The method validation results proved that the method is precise, accurate, and linear and could be applied in combination with autoclaving treatment for sample preparation in estimation of N-nitrosamines in meat for monitoring food safety.