N E W S L E T T E R F R O M TO S H V I N A N A LY T I C A L P V T. LT D. JULY 2013

Transcription

1 N E W S L E T T E R F R O M TO S H V I N A N A LY T I C A L P V T. LT D. JULY 213

2 Analysis of Residual Pesticides in Foods Using Twin Line GC MS To obtain highly reliable data from analyzing residual pesticides in foods by GC requires confirmation tests using columns with different liquid phases. In contrast, with GC- MS, confirmation is possible by using not only retention times, but also m/z values. Therefore, normally only one type of column is used for tests. However, due to the increased number of pesticides and the diversification in substances subject to inspection, users are demanding higher reliability by using columns with different liquid phases, even for GC-MS analysis. Therefore, a Twin Line MS Kit, which enables the installation of two types of columns with different liquid phases in one MS, without the use of splitters or flow restrictors, was used to analyze residual pesticides in food. Experiment Test Solution Preparation Method To evaluate the effectiveness in analyzing actual samples, test solutions of six agricultural products: spinach, carrots, cabbage, brown short-grain rice, oranges, and apples, were prepared in accordance to the test method specified by the Japanese Ministry of Health, Labour, and Welfare Method of Simultaneous Testing for Pesticides and Other Components (in Agricultural Products) [1]. Pesticides were then added to these test solutions to a concentration of.1 µg/ml. Analytical Conditions for Twin Line GC-MS The following analytical conditions were used to measure the test solutions. Columns 1 and 2 each had a dedicated sample vaporization chamber, each of which was used alternately for analysis. Twin Line MS System Connecting two different columns to the MS unit at the same time allows the smooth acquisition of application data with different columns, without shutting off the MS vacuum. The outlets of the two columns were connected directly to the mass spectrometer interface, without using flow restrictors. Therefore, the same retention times and retention indices can be used as in methods for a single column. Due to the lack of losses for adsorption or other factors and due to the high-capacity differential vacuum system, the same sensitivity levels can be obtained as for a single column. Results and Discussion Results and Discussion Column Selection GCMS - QP 21 Ultra When two types of columns are used elution patterns must be different Therefore it is reasonable to combine a lowpolarity column with a middle polarity column In addition since two columns are installed in the same column oven the maximum operating temperature capacity must be at least 3 C for both columns (maximum temperature forheating parameters) Therefore based on results previously reported [2] Rtx-5MS (33/35 C and Rtx-OPPesticides 2 (31/33 C columns were selected GC-MS Column1 Column2 : GCMS-QP21 Ultra (Twin Line MS kit) : Rtx-5MS (3 ml. x.25 mmi.d.,.25 µm) : Rtx-OPPesticides2 (3 ml. x.25 mmi.d.,.25 µm) [GC] Vaporization chamber temperature : 25 C Column oven temperature : 5 C (1 min) -> (25 C /min) -> 125 C -> (25 C /min) -> 3 C (15 min) Injection mode : Splitless (Sampling time: 1 min) High pressure injection : 25 kpa (1.5 min) Carrier gas : Helium Control mode : Linear velocity (47.2 cm/sec) Injection quantity : 2. µl [MS] Interface temperature : 25 C Ion source temperature : 2 C Measurement mode : Scan Mass range : m/z 5-46 Event time :.5 sec Emission current : 6 µa (normal) 2 JULY 213

3 Overlap Between Added Pesticides and Impurities Figures 1 and 2 show examples of how the overlap between pesticides and impurities differs for the two columns. In Figure 1 results for Rtx-OPPesticides2, Fenvalerate-2 is affected by impurities, but for Rtx-5MS, it is not affected by impurities. In Figure 2 results for Rtx-5MS, Triadimenol-1 is affected by impurities, but for Rtx-OPPesticides2, it is not affected by impurities. Fig. 1: Mass Chromatogram of Orange Extract Spiked with Fenvalerate-2 (left: Rtx-5MS; right: Rtx-OPPesticides2) Fig. 2: Mass Chromatogram of Brown Rice Extract Spiked with Triadimenol-1 (left: Rtx-5MS; right: Rtx-OPPesticides2) Verifying Detection of Pesticides in Actual Samples For Rtx-5MS, an impurity peak coexists at the retention time for captan, as shown in Figure 3, which makes it difficult to determine whether or not captan is present. However, for Rtx-OPPesticides2, there are no impurity peaks coexisting at the retention time for captan, which makes it easy to determine that the pesticide was not detected. Fig. 3: Mass Chromatogram of Brown Rice Extract at the Retention Time for Captan (left: Rtx-5MS; right: Rtx-OPPesticides2) References [1] Test method specified by Japanese Ministry of Health, Labour, and Welfare; [2] By Ueno, Ohshima, Saito, Matsumoto; Journal of the Food Hygienic Society of Japan 41, (21) JULY 213 3

4 Measurement of Residual Solvents in Pharmaceuticals by Headspace GC - USP <467> Residual Solvents - Procedure A - Residual solvents in pharmaceuticals are defined as volatile organic compounds used in or generated from the manufacture of drug substances, pharmaceutical additives, or drug products. They are strictly controlled according to risk classifications from Class 1 to Class 3, which are based on the risk to human health. Headspace GC methods specified in the USP (U.S. Pharmacopeia), General Chapters <467> Residual Solvents, are commonly used for analysis of residual solvents. These USP methods were created based on the analytical methods specified in the EP (European Analysis Conditions Analysis Conditions HS-2 Results 1. Class 1 Pharmacopoeia), in accordance with policies specified by the ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use). This Application Data Sheet presents data obtained using the Shimadzu HS-2 Headspace Sampler and Shimadzu GC-21 Plus Gas Chromatograph, from Class 1 and Class 2 standard solutions, in accordance with Water- Soluble Articles, Procedure A, in USP <467> Residual Solvents. Oven Temp. : 8 C Shaking Level : Off Equilibrating Time : 6 min Sample Pressurization : 75 kpa Pressurizing Time : 1 min Load Time :.5 min Injection Time : 1 min Needle Flush Time : 2 min Sample Line Temp. : 11 C Transfer Line Temp. : 12 C Vial Capacity : 2 ml GC-21 Plus Column : Rxi-624SilMS 32 mm X 3 m, Split Ratio : 1:5 d.f. = 1.8 um Hydrogen : 4 ml/min Column Temp. : 4 C (2 min) 1 C/min Air : 4 ml/min 24 C (2 min) Carrier Gas Linear Velocity : 35 cm/sec (helium) FID Temp. : 26 C Makeup Gas : 3 ml/min (helium) Results GC - 21Plus with HS - 2 Figure 1 shows the Class 1 standard solution chromatogram. Procedure A requires that the S/N ratio obtained for 1,1,1- Trichloroethane in this chromatogram be 5 or higher. As shown, the S/N ratio was 2. Even for carbon tetrachloride, which had the lowest sensitivity level, the S/N was 1. S/N Ratio 1 1,1-Dichloroethene ,1,1-Trichloroethane 2 3 Carbontetrachloride 1 4 Benzene ,2-Dichloroethane 5 Fig. 1: Water-Soluble Articles, Procedure A, Class 1 Standard Solution Chromatogram 4 JULY 213

5 2. Class 2 Due to the large number of components in the Class 2 standard solution, it was separated into two mixtures: A and B. Respective measurement results are shown in Figures 2 and 3. Procedure A requires that the resolution for acetonitrile and methylene chloride in the Class 2 standard solution Mixture A chromatogram be 1. or greater. Figure 4 shows that, using the Restek Rxi-624SilMS low-bleed column, the specified peaks are completely separated, with a resolution of 1.5. Fig. 2: Water-Soluble Articles, Procedure A, Class 2 Mixture A Standard Solution Chromatogram Fig.4: Separation Between Acetonitrile and Methylene Chloride 1 Methanol 2 Acetonitrile 3 Methylene chloride 4 trans-1,2-dichloroethene 5 cis-1,2-dichloroethene 6 Tetrahydrofuran 7 Cyclohexane 8 Methylcyclohexane 9 1,4-Dioxane 1 Toluene 11 Chlorobenzene 12 Ethylbenzene 13 m+p-xylene 14 o-xylene The area repeatability (RSD%) was evaluated by measuring the sample 2 consecutive times. The resulting RSD % value was between 1 % and 3 %, which indicates a higher repeatability than obtained using previous headspace samplers (see Table 1). The HS-2 headspace sampler achieves this unprecedented high repeatability by maintaining a uniform temperature distribution within the air tank oven and by using an advanced pressure control (APC) system for precise pressure control. Fig. 3: Water-Soluble Articles, Procedure A, Class 2 Mixture B Standard Solution Chromatogram Table 1: Peak Area Repeatability of Class 2A & 2B 1 Hexane 2 Nitromethane 3 Chloroform 4 1,2-Dimethoxyethane 5 Trichloroethene 6 Pyridine 7 Methylbutylketone 8 Tetraline JULY 213 5

6 Validation of BioLumix Rapid Microbiology System Against USP Methodology Abstract To meet the challenges of the new FDA microbiological regulations requires simpler, faster, and more streamlined tests. These tests have to comply with the methodology described in the relevant USP chapters. The BioLumix system, as any other alternative testing system, has to be validated against the new chapters of USP (<221>, <222>, and <223>). This poster describes the validation protocol used. The validated BioLumix system allows companies to greatly reduce the amount of time and money required to have their samples tested by independent outside laboratories. This fully automated system delivers much faster results generating an automated certificate of analysis within 48 hours. Consequently, it enables earlier release of raw materials and finished products while adding profits to a company's bottom line. Introduction Introduction Nutraceutical and dietary supplement cgmps require that manufacturers take steps to ensure products are free of contamination with objectionable microorganisms. Objectionable organisms are organisms that might jeopardize the safety of the products or organisms that are able to grow in the product, affecting its stability. Therefore, manufacturers need to test raw materials and finished products for relevant microorganisms. Guidelines and considerations to help manufacturers determine the appropriate test(s) are found in chapter <223>. The actual quantitative and qualitative testing procedures are found in chapters <221> and <222>. The actual official ruling on dietary supplement cgmps (21CFR Part 111) can be found in chapter <275>. Even the facilities that are subcontracting their microbiology testing need to become very familiar with these chapters, since many outside laboratories are utilizing BAM or AOAC methodology rather than the USP methodology. Understanding the appropriate testing procedures and the results obtained for a specific product will dictate how and when the manufacturer should release product and issue recalls if needed. Under the current edition for Nutraceutical, in addition to TAMC and TYMC, chapter <221> includes Enterobacterial Count (bile-tolerant Gram negatives), whereas chapter <222> covers tests for S. aureus, Salmonella species, E. coli and Clostridia. USP recommends microbial limits for different types of raw materials and finished products. As an example, for dried 6 or powdered botanicals, the recommended limit for TAMC 5 is 1 CFU/g, whereas for TYMC and Enterobacterial 3 Count the recommended limit is 1 CFU/g. Salmonella and E. coli must be absent in 1 grams from this product. A common practice in industry is to test only for TAMC and TYMC to reduce costs. However, it must be realized that these tests do not reveal any indication as to what type of microorganism might be growing in the raw material or finished product. The new required Enterobacterial count test will help to predict the presence of pathogens such as E. coli or Salmonella but it will not provide any indication of S. aureus or Clostridia. Nevertheless, this test is a limit test, which means that the product can still contain Enretobacteriaceae within a specified range; so, without an additional pathogen specific test, a single colony of E. coli or Salmonella could go unrecognized. It is important to consider that raw materials can be a primary source of product contamination with objectionable microorganisms. Ingredients of animal or botanical origin that are not highly processed, or that have an unpredictable level of microbiological contamination, require more frequent testing than synthetic or well characterized products. The quality of finished product with respect to microbial growth should never be undervalued and maintaining appropriate microbiological product attributes is crucial. It is always better to spend a few extra dollars on additional testing rather than paying for it in lawsuits or recalls. The System The System The BioLumix system (Figure 1) is comprised of an instrument with a capacity for testing 32 individual assay vials, software, and disposable vials. Each instrument serves as an incubator. Up to 32 instruments can be connected to one computer. The software enables rapid, real-time results to be transferred to where they are needed most, without any operator involvement. The available disposable vials include: total aerobic count (TAC), Yeast and Mold (YM), Enterobacteriaceae (ENT), E. coli (EC), Pseudomonas (PSE), Staphylococcus (STA) and Salmonella (SAL). All assay vials are provided with a ready to use sterile media and a certificate of analysis. JULY 213

7 Figure 1: The System Figure 2: Disposable Vials Definitions cgmps: Current Good Manufacturing Practices IQ : Installation Qualification: Providing documented evidence the system is installed as specified by the manufacturer in the location where it will be operated. OQ : Operation Qualification: Providing documented evidence the system operates as specified by the manufacturer when installed in its intended location. PQ : Performance Qualification: Providing documented evidence the system performs as expected when testing is performed using the media and test substrates that will be used for testing using this system. Specificity : The ability of the test method to detect the range of organisms that may be present in the test article. Limit of Detection: The lowest number of microorganisms in a sample that can be detected under the stated experimental conditions. False Negative : False negative tests are those which are inoculated and generate a positive result in the reference method but are negative in the test method. If this occurs at a certain inoculation level then it is referred to as the Limit of Detection. False Positive : False positive tests are those tests that are not inoculated with the target organism but generate a positive result in the test method but not in the reference method. Time to Detection : Time it takes for a positive signal on the BioLumix or to a positive result in the reference method. Repeatability : The ability to get consistent results when a number of samples are analyzed. Ruggedness : The degree of precision of test results obtained by analysis of the same samples under a variety of normal test conditions, such as different analysts, instruments or reagent lots. Validation Elements Installation Qualification : Includes major component identification and validation, validation of environmental conditions, electrical requirements, computer qualification, installation checklist, and calibration documentation. Operation Qualification : Includes a unique SOP for all product assay combinations that need to be performed, training documents, software characteristics, certification and verification of 21 CR part 11 compliance, as well as an operation checklist. Performance Qualification : In the heart of the validation package. It includes: Side-by-side comparison of the BioLumix data to USP methodology. This comparison is done individually for each assay. For example for Total Aerobic Count 159 samples were analyzed for total aerobic count at various specification levels. 113 samples were below spec by both methods, while 46 were above the specified level by both methods. There was 1% agreement between the two methods. For Yeast and Mold Count 142 samples were analyzed for yeast and mold at various specification levels. 18 samples were below spec by both methods, while 32 were above the specified level by both methods. There were two samples below the specified level by the BioLumix method and just above the specified level by plates. There was 98.6% % agreement between the two methods. Similar comparisons were conducted for Enterobacteriaceae, E. coli, S. aureus, and P. aeruginosa. Specificity (also call inclusivity and exclusivity): For each of the different vials the specificity was determined. For Total aerobic count 8 well diverse bacteria were inoculated into the TAC vial. All organisms tested detected by the TAC vials. For Enterobacteriaceae, 29 bacteria belonging to the Enterobacteriaceae genus were tested. All detected in the vial. 18 bacteria strains that do not belong to the genus Enterobacteriaceae were also tested in the Enterobacteriaceae vial and none of them detected in the vial. 1 E. coli strains were tested in the E. coli vial, and all detected in the vial. 37 bacteria strains that are not E. coli were also tested and none detected in the vial. All other vials were tested. Detection Limit : The limit of detection for the BioLumix system is equal or better than the plate count method and is close to one viableorganism capable of growth in the JULY 213 7

8 medium. In several cases one organism was detected in the vial, and not in the plates. With levels of 1-2 colonies/ to a vial or a plate the BioLumix was able to detect their presence in 16/2 cases while the plate count method was only capable of detecting 12/2. The variability of the vials was equal or better than the plate count method. Repeatability : The standard deviation for each of the media was determined using naturally contaminated samples and samples inoculated with various organisms. Figure 3 shows reproducibility data Robustness : Three parameters were used to test the robustness of the BioLumix system - The effect of changes in the instrument incubator temperature, the effect of sample size, and the effect of medium volume in the vial. The robustness was tested in various media with several repetitions. The BioLumix system appears to be very robust and remain unaffected by small deliberate variations in method parameters. It shows high precision of test results obtained by analyzing the same microorganisms under a variety of different conditions. Figure 3: Repeatability data Ruggedness : Three parameters were used to test the ruggedness of the BioLumix system - Effect of Analyst, effect of unit (instrument), and effect of reagent lots. Two analysts tested two different media (total aerobic count and E. coli medium) with five diverse types of bacteria in each (a total of 1 paired data sets of 4 vials each). The data was presented and an ANOVA analysis shows that there was no significant difference in results among the analysts. Two different instrument were used with two different media (total aerobic count and Enterobacteriaceae medium), with four organism per media. The data was presented and an ANOVA analysis shows that there was no significant difference in results among the machines utilized. Three different lots of each medium were tested in this study. Three types of media (total aerobic count, coliform medium and E. coli medium), were tested each with four different bacteria. The data was presented and an ANOVA analysis shows that there was no significant difference in results among the lots of media. Therefore, the BioLumix system appears to be very rugged with high precision of test results obtained by analyzing the same microorganisms under a variety of different conditions. False Negative : A false negative is a test in which samples that are inoculated or naturally contaminated generate a positive result in the reference method (plate count) but are negative in the test method (BioLumix). Two naturally contaminated (wild Yam and Spirulina products) were used, each was subdivided into 1 samples of 1 gr. and tested by the plate count method and the BioLumix dilute to spec approach. Total count: Two levels of specifications (1, cfu/gr. and 1, cfu/gr.) were tested for each product. The BioLumix procedure detected correctly all 2 sample combinations as being above the specified level. In one case the plate count method showed a count below the spec level although the remaining 9 samples of the same lot were above the specified level. Yeast and mold : two levels of specifications (1 cfu/gr. and 1 cfu/gr.) were tested for each product. Both methods correctly detected that the samples were above the specified level. The BioLumix system had no false negative results. False Positive : A false positive is a test in which a sample that is not inoculated with the target organism generates a positive result in the BioLumix system but not in the Plate Count method. Two assays (Total aerobic count and yeast and mold) were used to test the false negative rate of the BioLumix system. The total count yielded a 2% false positive rate while the yeast and mold yielded % false positive rate. Conclusion Conclusion The BioLumix System was validated against the methodology described in USP <221>, <222>, and <223>. The validation included Installation Qualification, Operation Qualification, with especial emphasis on Performance Qualification. The data shows the equivalency of the BioLumix system to the USP methodology. The BioLumix all-in-one rapid automated system is the most advanced system of its kind offering simplified microbiology with an automated certificate of analysis in 48 hours. 8 JULY 213

9 Analysis of Sodium & Potassium in Fatty Acid Methyl Esters (FAME) Introduction In FAME, Sodium (Na) and Potassium (K) may be present due to the presence of catalysts used during its' production. Excessive amounts of Na and K may form abrasive solids or metallic soaps and lead to formation of deposits on the injectors and valves and cause clogging of (1) filter. Hence, most countries like Europe, in EN14214 specify that the total of Na and K, or also known as Group I Metals, must not exceed 5 mg/kg when combined. According to EN14214, Na and K can be determined using flame atomic absorption spectrophotometry (AAS). The (2) (3) test methods used are EN1418 and EN1419. In this article, the analysis of Na and K in FAME prepared from palm olein and coconut oil using flame AAS is shown. Materials & Methods The 1 ppm multi-organometallic standard (S-21) and Element Blank Oil-75 Viscosity were from Conoco Phillips Inc, USA whereas xylene was from JT Baker, USA. The FAME samples were produced from palm olein and coconut oil. All the standard solutions and samples were prepared in xylene according to EN1418 and EN1419 for the analysis of Na and K respectively. Na and K were analysed with a Shimadzu AA-63 flame AAS using the analytical conditions as shown in Table 1. Table 1. Analytical conditions for Na and K. Results AA - 7 The Na and K calibration curves are shown in Figures 1 and 2 below. As could be seen, both calibration curves have good linearity with a regression factor of more than.999. Figure 1. Na calibration curve. Conditions Na K Wavelength 589. nm nm Slid width.2 nm.7 nm Lamp Current 12 ma 1 ma Lamp mode Air-C H 2 2 Flame No background correction Air : 17. L/min C H : 1.4 L/min 2 2 Burner height 8 mm 7 mm Air : 17.5 L/min C H : 1.5 L/min 2 2 Figure 2. K calibration curve. JULY 213 9

10 (5) To determine limit of detection (LOD), the following formula was used: LOD = 1 x SD of blank of 2 repetitions x VF slope of calibration curve where SD : Standard deviation VF : Volume of sample WF : Weight of sample WF The LOD for Na and K analysis was.7 mg/kg and.1 mglkg respectively. The Na and K content in the two FAME samples are shown in Table 2 below. Table 2: Na and K content in FAME samples. As this is a preliminary study, we consider that matrix interference is not present if the %R is between 9-11%. From the results in Table 3, the % R was more than 9% for both Na and K analysis. Hence, most likely there is no matrix interference in the analysis. Table 3: Matrix interference test Sample FAME from Palm Olein FAME from Palm Olein spiked with.125 ppm Na % Recovery = 94.6%.1 ppm.98 ppm Measured (1) FAME from Palm Olein Na Content K Content FAME from Coconut Oil.3 ppm.4 mg/kg (<LOD of.7 mg/kg).2 mg/kg (<LOD of.1 mg/kg) FAME from Coconut Oil spiked with.125 ppm Na.99 ppm Group I Metals content <.17 mg/kg (2) FAME from Coconut Oil Na Content K Content.7 mg/kg.7 mg/kg (<LOD of.1 mg/kg) Group I Metals content <.17 mg/kg For these two FAME samples, the Group 1 Metals content was less than 5 mg/kg and hence, within the EN14214 specifications. The FAME samples were diluted in xylene before analysis. To determine whether there are any matrix interferences which can affect the accuracy of an analysis, the diluted sample was spiked with a known amount of standard solution. The percentage recovery (%R) is calculated (6) according to the formula belowc : %R = [ (SSR - SR) I SA] x 1% where SSR : Spiked sample result SR : Sample result SA : Spike added % Recovery = 93.7% Conclusions Flame AAS is a convenient and accurate method to measure Na and K in FAME prepared from palm olein and coconut oil. References (1) A biodiesel primer ( (2) BS EN 1418:23 Fat and oil derivatives - fatty acid methyl esters (FAME) - determination of sodium content by atomic absorption spectrophotometry. (3) BS EN 1419:23 Fat and oil derivatives - fatty acid methyl esters (FAME) - determination of potassium content by atomic absorption spectrophotometry. (4) BS EN 14214:23 Automative fuels - fatty acid methyl esters (FAME) for diesel engines - requirements and test methods. (5) SAP/CSC/CAM/AAS-31 - How to determine LOD in AAS. (6) USEPA Contract Laboratory Program - Statement of work for inorganic analysis. 1 JULY 213

11 Highest sensitivity in its class by unique ion optics system Fast GCMS analysis with high speed scanning of 2, amu/sec and high speed data acquisition High capacity Differential Vacuum System facilitates use of Wide bore capillary column with 15 ml/min flow rate ASSP-Advanced Scanning Speed Protocol function ensures utmost sensitivity even with higher scanning speed Enhances productivity with minimal downtime by Easy stop feature Saves costly carrier gas by unique Ecology mode JULY

12