Analysis Of United States Pharmacopoeia (USP . - PerkinElmer

APPLICATION NOTE Gas Chromatography AUTHORS The data presented here demonstrate that Tracy biochemical Dini HTRF and cellular assays PerkinElmer, Inc., Shelton, CT USA offer a comprehensive and easy-toLee Marottaplatform for the identification implement PerkinElmer, Inc., Shelton, CT USA of potent biotherapeutics to address Miles Snow pathologies. autoimmune PerkinElmer, Inc. Woodbridge, ON Canada Analysis of United States Pharmacopoeia (USP) Grade Alcohol (Ethyl alcohol) Impurities Introduction Alcohol (Ethyl alcohol) or Ethanol, is a chemical widely used in pharmaceuticals and consumer products. Ethanol is used in formulations of products like skin disorder treatments, over-the-counter (OTC) products, cosmetics and beauty products, and disinfectants and hand sanitizers. It is important to monitor the quality of ethanol used in these products to protect human and animal health. This application note analyzes trace level impurities that can be found in ethanol as per USP guidelines. The first section of this note describes the analysis of USP grade Ethanol as per USP recommended condition and procedures. The second section describes a modified procedure that produces excellent results with a greatly improved data throughput and significant reduction in laboratory sample preparation time.

Analysis of United States Pharmacopoeia (USP) Grade Ethanol Impurities Instrumentation Instrumentation The PerkinElmer Clarus 690 GC with integrated autosampler was used to perform these experiments. The Clarus 690 GC was configured with a capillary injector and dual wide range flame ionization detector (FID). A PerkinElmer Elite 624 and BAC-1 columns were installed in the injector via a two-hole ferrule or a “Y” splitter as shown in Figure 1. The GC conditions required for the analysis are listed in Table 1. The GC conditions, following the USP protocol, are found in Table 1. A faster method was developed by PerkinElmer as an alternative using the same columns as shown in the Alternate Method section later in this application note. Table 1: Chromatography conditions GC Parameters Instrument PerkinElmer Clarus 690 GC Carrier Gas Helium Columns Column Pneumatics Elite-624 30 m 0.32 mm 1.8 µm N9316203 Elite-BAC1 30 m 0.32 mm 1.8 µm N9315071 LCV: 35 cm/sec Split flow: 20 mL/min Autosampler Parameters Syringe size 5 µL Injection volume 1 µL Injection speed Normal # of plunges 8 Pre-washes 0 Sample washes 2 Post washes 4 Viscosity delay 0 Injector Parameters Injector Type: CAP Temp: 200 C Detector Parameters Type WR-FID Temperature 230 C Range 20 Att -6 Hydrogen 30 mL/min An aliquot of the Ethanol sample is added to a 2 mL autosampler vial and placed into the integrated autosampler of the Clarus 690 Gas Chromatograph (GC). Air 450 mL/min Data rate 12.5 pt/sec Standard Preparation Oven initial temperature 40 C The USP method of standardization for Ethanol utilizes an uncommon technique of standard addition. The advantage of the method is accurate results and no high purity solvents are required, the concept is to spike the sample with a known amount of target analyte and compare this peak area with the peak area of an unspiked sample. Benzene, for example: Oven initial hold 12 minutes Ramp Rate 10 C/minute Final temperature 240 C Final time 10 minutes Oven maximum 260 C Equilibration time 0 Figure 1: 30 m 0.32 mm Elite 624 and Elite-BAC columns used in this study. Experimental Sample Preparation Oven Parameters Benzene Calculation Data Acquisition Result [BE/(BT- BE)] x CB Data acquisition and data processing was performed using TotalChrom chromatography data system (CDS) software. BE Peak area of benzene from Sample solution A BT Peak area of benzene from Standard solution D CB Concentration of benzene in Standard solution D (µL/L) www.perkinelmer.com 2

Analysis of United States Pharmacopoeia (USP) Grade Ethanol Impurities Calibration Ethanol Each sample requires 6 chromatographic runs as follows Sample solution A: Sample solution B: Standard solution A: Standard solution B: Standard solution C: Standard solution D: Alcohol (substance under test) 300 uL/L of 4-methylpentan-2-ol in Sample solution A 200 uL/L of methanol in Sample solution A 10 uL/L of methanol and 10 uL/L of acetaldehyde in Sample solution A 30 uL/L of acetal in Sample solution A 2 uL/L of Benzene in Sample solution A The USP method requires different calculation equations for each analyte target and the sample passes if the result is “Not More Than” (NMT) the concentrations listed for Standard solutions above. Methanol Calculation Result (Ru/ Rs) Ru Peak area of methanol from Sample solution A Rs Peak area of methanol from Standard solution A Benzene Calculation Result [BE/(BT- BE)] x CB BE Peak area of benzene from Sample solution A BT Peak area of benzene from Standard solution D CB Concentration of benzene in Standard solution D (µL/L) Acetaldehyde Calculation (sum of acetaldehyde and acetal) Result {[AE/(AT-AE)] x CA} {[DE/(DT-DE)] x CD x (MR1/MR2)} AE Peak area of acetaldehyde from Sample solution A AT Peak area of acetaldehyde from Standard solution B CA Concentration of acetaldehyde in Standard solution B( µL/L ) DE Peak area of acetal from Sample solution A DT Peak area of acetal from Standard solution C CD Concentration of acetal in Standard solution C ( µL/L ) MR1 Molecular weight of Acetaldehyde, 44.05 MR2 Molecular weight of Acetal, 118.2 Any other impurity Calculation Result (Ru/Rm) x Cm Ru Peak area of each impurity in Sample solution B Rm Peak area of 4-methylpentan-2-ol in Sample solution B Cm Concentration of 4-methylpentan-2-ol in Sample solution B (µL/L) Results and Discussion Since there are many organic compounds, and several co-elute, dual column confirmation was used to prevent the reporting of false negatives, as suggested by USP for the conformation of benzene. The forensic industry uses this technique for blood alcohol investigation to confirm compound identification. The USP method uses a G43 (common name 624) phase as the reporting column for results and allows for a column of different polarity stationary phase for the confirmatory column. The BAC 1 phase was chosen for its chromatographs and ability to effectively separate these compounds. In addition, it is well vetted by the forensics industry as the reporting column for blood alcohol investigation. Ethanol was run spiked and unspiked as shown below in Figure 2 to 7. Figure 2: Ethanol sample under test. www.perkinelmer.com 3

Analysis of United States Pharmacopoeia (USP) Grade Ethanol Impurities Figure 3: 300 µL/L 4-Methylpentan-2-ol spike. Figure 4: 200 µL/L Methanol spike. Acetaldehyde Ethanol Unknown Methanol Ethanol Figure 5: 200 µL/L Methanol and Acetaldehyde spike. www.perkinelmer.com 4

Analysis of United States Pharmacopoeia (USP) Grade Ethanol Impurities Acetal Figure 6: 30 µL/L Acetal spike. Benzene Figure 7: 2 µL/L Benzene spike. An Ethanol sample of unknown history easily passed the USP criteria as show in Table 2. Table 2: Calculation of impurities in Ethanol. Name Acetaldehyde Methanol Benzene Acetal 4-methyl-2-pentanol area [µV·s] area [µV·s] area [µV·s] area [µV·s] area [µV·s] Ethanol Blank A 0 0 3.1 0 0 300 ppm 4-methyl-2-pentanol 0 0 0.8 0 2763.4 200 ppm methanol 0 336.1 4.3 0 0 10 ppm methanol/acetaldehyde 96 15 2.3 0 0 30 ppm acetal 0 0 1.5 164.4 0 2 ppm benzene 0 0 22.4 0 0 0 Ethanol Blank B 0 0 0.7 Acceptance Limit (NMT) 10 200 2 Ethanol Sample A results 0 0 0.32 www.perkinelmer.com 0 300 0 5.54 5

Analysis of United States Pharmacopoeia (USP) Grade Ethanol Impurities Table 3: USP impurities in Ethanol, action limits (NMT) and repeatability. Ethanol Compound Action Limit (µL/L) Precision % RSD Acetaldehyde 10 1.3 Methanol 200 1.3 Benzene 2 1.9 Acetal 30 1.3 4-Methyl-2-Pentanol 300 1.4 Summary As we have demonstrated in this application, the PerkinElmer dual column FID Clarus 690 GC was able to easily analyze impurities in Ethanol using the USP method with excellent accuracy and selectivity for alcohols used for manufacturing hand sanitizer by USP methodology. Alternate Method Alternate Introduction The analysis of Ethanol by the USP method as described above works well with good results. There are a few items that could be improved upon to increase lab productivity. Alternate Experimental The Instrument configuration is identical as described vide supra. The chromatography column and condition are similar to the above official USP conditions and are only modified for improved data quality and speed of analysis. The resulting modified parameters are highlighted below in Table 4. Table 4: Alternate instrument parameters. GC Parameters Instrument PerkinElmer Clarus 690 GC Carrier Gas Helium Columns Column Pneumatics Elite-624 30 m 0.32 mm 1.8 µm N9316203 Elite-BAC1 30 m 0.32 mm 1.8 µm N9315071 LCV: 35 cm/sec Split flow: 20 mL/min Autosampler Parameters Syringe size 5 µL Injection volume 1 µL Injection speed Normal # of plunges 6 Pre-washes 2 Sample washes 2 Post washes 2 Viscosity delay 0 www.perkinelmer.com Table 4: Alternate instrument parameters. Injector Parameters Injector Type: CAP Temp: 200 C Detector Parameters Type WR-FID Temperature 230 C Range 20 Att -6 Hydrogen 30 mL/min Air 450 mL/min Data rate 12.5 pt/sec Oven Parameters Oven initial temperature 40 C Oven initial hold 5 minutes Ramp rate 70 C/minute Final temperature 230 C Final time 1 minute Oven maximum 230 C Equilibration time 0 Alternate Method Results and Discussion Alternate method standard combined all individual standards into a combined stock standard as shown in Figure 9. External single point standardization was used on all components and results were calculated using the USP calculations, vide supra. Figure 8 shows an unknown Ethanol that was processed and the results are shown in Figures 10 and 11. 6

Analysis of United States Pharmacopoeia (USP) Grade Ethanol Impurities Figure 8: Ethanol test sample under alternate conditions. USP impurities in ethanol in less than 7.5 minutes Figure 9: USP impurities Standards at Action Limits. Ethanol A Combined standard Repeatability and RSD Figure 10: Combined Standard G43 column. www.perkinelmer.com 7

Analysis of United States Pharmacopoeia (USP) Grade Ethanol Impurities Ethanol B Combined standard Repeatability and RSD Benzene Figure 11: Combined Standard BAC-1 column. Table 6: % RSD of ethanol impurities. Ethanol Compound Precision % RSD (n 8) Acetaldehyde 0.78 Methanol 1.17 Benzene 1.32 Acetal 0.97 4-Methyl-2-Pentanol 0.92 If benzene is detected, it is required to rerun the sample with a different polarity column to confirm the peak is indeed benzene, not a coeluting nontarget. The configuration used in this study analyzes all target through each column sets thus eliminating the need to remove columns and/or rerunning samples with positive benzene results. Figure 12 shows benzene on two different polarities columns. Benzene The G43 Column Benzene The Confirmatory Column Figure 12: Benzene confirmation on G43 vs BAC-1. www.perkinelmer.com 8