Productivity Improvements for Dual Column Applications: USP <467> and Blood Alcohol Analysis Rebecca Veeneman, PhD Applications Chemist Agilent Technologies
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A simplified approach to dual column analyses USP <467> and Blood Alcohol Analysis
Dual Column Configuration Headspace to SSL Inlet splitter flow chip equally splits to two columns Two columns to two FID detectors Inlet FID 1 FID 2 Guard Chip Inlet splitter flow chip Column 2 Column 1
USP <467> Residual Solvent Requirements Analysis of residual solvent is a critical application in the pharmaceutical industry. The choice of solvent during manufacturing can improve yield or affect chemical properties of the product synthesized. Solvents do not enhance the product s efficacy and must be removed as completely as possible to meet product specification and good manufacturing practices. USP467 specifies a single column analysis A secondary analysis is performed if the solvent is found above limit detection. With an Intuvo 9000 GC configured with an inlet splitter both analyses can be performed in one analytical run. USP 40, general chapter USP <467> Residual Solvents https://hmc.usp.org/sites/default/files/documents/hmc/gcs-pdfs/c467.pdf
USP <467> Residual Solvent Methods Class 1, Class 2A, and Class 2B residual solvents were prepared at their limit concentrations in water. Class 1 (5190-0490) was prepared in the following manner: 1. 1.0 ml stock and 9 ml of DMSO was diluted to 100 ml with water 2. 1.0 ml from step 1 was diluted to 100 ml with water 3. 10 ml from step 2 was diluted to 100 ml with water Class 2A (5190-0492) was prepared by diluting 1 ml of stock to 100 ml with water Class 2B (5190-0513) was prepared by diluting 1 ml of stock to 100 ml with water Headspace vials were prepared with 1 ml of each solvent and 5 ml of water. Samples were prepared in 10 ml HS vials
7697 Headspace parameters 7697 Headspace Sampler Setpoint Oven 85 C Loop 85 C Transfer line 100 C Vial equilibration time Injection duration Vial size Vial shaking Vial fill mode Vial fill pressure Loop ramp rate Loop final pressure Loop equilibration time 40 min 0.5 min 10 ml Level 2, 25 shakes/min Default (50 ml/min to 15 psi (0.1 min)) 15 psi 20 psi/min 0 psi 0.05 min
Intuvo Parameters Intuvo 9000 GC Oven Setpoint 40 C (5 min) 15 C/min to 180 C (3 min) Split/Splitless Inlet Split 5:1, 140 C DB-624 Select UI (123-0334UI-INT) 30m x 320µm x 1.8µm DB-WAX UI (123-7032UI-INT) 30m x 320µm x 0.25µm Constant flow 2 ml/min Controlled by column 1 FID (Front and Back) 250 C H2 Air 30 ml/min 400 ml/min N2 (makeup) 25 ml/min Jumper chip 140 C Bus 200 C Front/Back Signal 20 Hz Jumper chip set to the inlet temperature Bus temperature set to default
1.795 5.380 2.879 7.246 8.873 1.912 8.208 peak@8.208min 10.280 12.350 12.310 13.702 Response [pa] 4.327 peak@4.327min 8.535 peak@8.535min 17.061 8.014 peak@8.014min 8.463 peak@8.463min Class 1, DB-624 Select UI x10 1 DFID_Mix1_01 FID3A 1. 1,1-dichloroethane 2. 1,1,1-trichloroethane 3. carbon tetrachloride 4. benzene 5. 1,2-dichloroethane 1.025 1.02 1.015 1.01 1.005 1 0.995 0.99 0.985 0.98 0.975 0.97 0.965 0.96 0.955 0.95 0.945 0.94 0.935 0.93 0.925 0.92 0.915 0.91 0.905 0.9 0.895 0.89 0.885 0.88 0.875 0.87 0.865 0.86 0.855 0.85 0.845 0.84 0.835 0.83 0.825 0.82 0.815 0.81 1 3 2 4 5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 Retention time [min]
1.888 15.136 Response [pa] 2.264 peak@2.264min 3.135 peak@3.135min 6.736 peak@6.736min 3.892 peak@3.892min Class 1, DB-WaxUI 1. 1,1-dichloroethane 2. 1,1,1-trichloroethane 3. carbon tetrachloride 4. benzene 5. 1,2-dichloroethane x10 1 1.0175 1.015 1.0125 1.01 1.0075 1.005 1.0025 1 0.9975 0.995 0.9925 0.99 0.9875 0.985 0.9825 0.98 0.9775 0.975 0.9725 0.97 0.9675 0.965 0.9625 0.96 0.9575 0.955 0.9525 0.95 0.9475 0.945 0.9425 0.94 0.9375 0.935 0.9325 0.93 0.9275 0.925 0.9225 0.92 0.9175 0.915 0.9125 0.91 0.9075 0.905 0.9025 0.9 0.8975 0.895 0.8925 0.89 0.8875 0.885 0.8825 0.88 0.8775 0.875 DFID_Mix1_01 FID4B 1 2,3 4 5 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 Retention time [min]
Class 1 Repeatability Compound DB-624 RT RSD DB-624 Area RSD DB-Wax RT RSD DB-Wax Area RSD 1,1-dichloroethane 0.031% 3.3% 0.074% 2.1% 1,1,1- trichloroethane carbon tetrachloride 0.026% 2.6% 0.037% 1.8% 0.11% 4.8% Coelutes with 1,1,1- trichloroethane Coelutes with 1,1,1- trichloroethane benzene 0.017% 2.2% 0.055% 1.5% 1,2-dichloroethane 0.016% 3.9% 0.067% 1.8% Excellent RT repeatability for both columns Area RSD is 5% or better
2.902 peak@2.902min 8.459 5.238 peak@5.238min 5.747 peak@5.747min 7.746 peak@7.746min 9.662 peak@9.662min 10.801 7.344 peak@7.344min 9.499 peak@9.499min 12.716 peak@12.716min 8.093 peak@8.093min Response [pa] 10.653 peak@10.653min 12.199 peak@12.199min 12.326 peak@12.326min Class 2A, DB-624 Select UI 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 10.toluene 11.chlorobenzene 12.ethylbenzene 13.m-xylene 14.p-xylene 15.o-xylene x10 3 1.7 1.65 1.6 1.55 1.5 1.45 1.4 1.35 1.3 1.25 1.2 1.15 1.1 1.05 1 0.95 0.9 0.85 0.8 0.75 0.7 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 DFID_Mix2a_01 FID3A 1 2 3 4 5 6 7 8 9 13,14 10 12 15 11-0.05 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 Retention time [min]
1.895 3.747 peak@3.747min 5.156 peak@5.156min 6.644 peak@6.644min 17.220 2.413 peak@2.413min 2.949 peak@2.949min 4.888 peak@4.888min 8.717 peak@8.717min 7.784 peak@7.784min 9.254 peak@9.254min 2.251 peak@2.251min Response [pa] 6.054 peak@6.054min 8.028 peak@8.028min Class 2A, DB-Wax UI 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 10.toluene 11.chlorobenzene 12.ethylbenzene 13.m-xylene 14.p-xylene 15.o-xylene x10 3 1.275 1.25 1.225 1.2 1.175 1.15 1.125 1.1 1.075 1.05 1.025 1 0.975 0.95 0.925 0.9 0.875 0.85 0.825 0.8 0.775 0.75 0.725 0.7 0.675 0.65 0.625 0.6 0.575 0.55 0.525 0.5 0.475 0.45 0.425 0.4 0.375 0.35 0.325 0.3 0.275 0.25 0.225 0.2 0.175 0.15 0.125 0.1 0.075 0.05 0.025 0-0.025-0.05 DFID_Mix2a_01 FID4B 7 8 4 5 1 3,6 2 10 13 12 9 14 15 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 Retention time [min] 11
Class 2A Repeatability Compound DB-624 RT RSD DB-624 Area RSD DB-Wax RT RSD DB-Wax Area RSD methanol acetonitrile methylene chloride 0.23% 1.3% 0.22% 1.3% 0.023% 2.1% 0.039% 2.0% 0.018% 0.98% 0.033% 0.78% trans-1,2- dichloroethene 0.016% 0.85% 0.023% 0.72% cis-1,2- dichloroethene 0.012% 0.76% 0.039% 0.78% tetrahydrofuran cyclohexane methylcyclohexane 1,4-dioxane toluene chlorobenzene ethylbenzene m-xylene p-xylene o-xylene coleutes with 0.018% 0.95% methylene chloride coelutes with methylene chloride 0.011% 0.96% 0.013% 0.96% 0.0087% 0.98% 0.018% 0.96% 0.012% 1.9% 0.025% 1.7% 0.0073% 0.81% 0.029% 0.80% 0.0061% 0.69% 0.024% 0.69% 0.0060% 0.84% 0.016% 0.86% 0.0061% 0.82% 0.017% 0.82% coleutes with m-xylene coelutes with m-xylene 0.015% 0.83% 0.0059% 0.81% 0.026% 0.81% Excellent RT repeatability for both columns Area RSD is 2% or better
1.922 7.815 * peak@7.815min 8.473 * peak@8.476min 12.324 14.871 10.645 peak@10.642min 6.229 peak@6.230min 9.245 peak@9.247min 11.335 peak@11.334min Response [pa] 16.464 peak@16.463min Class 2B, DB-624 Select UI 1. n-hexane 2. nitromethane 3. chloroform 4. 1,2-dimethoxyethane 5. trichloroethane 6. pyridine 7. 2-hexanone 8. tetralin x10 1 3.3 3.25 3.2 3.15 3.1 3.05 3 2.95 2.9 2.85 2.8 2.75 2.7 2.65 2.6 2.55 2.5 2.45 2.4 2.35 2.3 2.25 2.2 2.15 2.1 2.05 2 1.95 1.9 1.85 1.8 1.75 1.7 1.65 1.6 1.55 1.5 1.45 1.4 1.35 1.3 1.25 1.2 1.15 1.1 1.05 1 0.95 0.9 0.85 0.8 0.75 DFID_Mix2b_01 FID3A 1 2 3 4 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 Retention time [min] 5 6 7 8
2.257 3.715 peak@3.705min 4.930 peak@4.930min 5.677 peak@5.676min 6.046 7.065 peak@7.065min 8.011 peak@8.012min 9.196 peak@9.194min 17.216 peak@2.028min Response [pa] 12.934 peak@12.936min Class 2B, DB-Wax UI 1. n-hexane 2. nitromethane 3. chloroform 4. 1,2-dimethoxyethane 5. trichloroethane 6. pyridine 7. 2-hexanone 8. tetralin x10 1 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1 0.9 0.8 0.7 DFID_Mix2b_01 FID4B 1 1.895 2.030 4 5 3 7 2 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 Retention time [min] 6 8
Class 2B Repeatability Compound DB-624 RT RSD DB-624 Area RSD DB-Wax RT RSD DB-Wax Area RSD hexane 0.021% 4.4% 0.039% 4.5% nitromethane 0.42% 4.9% 0.018% 2.5% chloroform 0.0099% 4.8% 0.026% 3.2% 1,2- dimethoxyethane 0.013% 3.6% 0.089% 3.6% trichloroethane 0.010% 3.4% 0.028% 3.1% pyridine 0.020% 1.7% 0.034% 1.7% 2-hexanone 0.0081% 2.7% 0.020% 0.74% tetralin 0.0078% 2.1% 0.016% 2.1% Excellent RT repeatability for both columns Area RSD is 5% or better
Blood Alcohol Concentration Application Requirements Determination of blood alcohol concentration requires rigorous control. Many labs use flame ionization detection (FID) which lacks identification capabilities Often a second system with a column having different retentive properties is used to confirm analyte identification Blood alcohol concentration determination and confirmation can be achieved simultaneously with the Intuvo 9000 GC. An Intuvo 9000 GC system was equipped with a 7697A Headspace Sampler and configured with an inlet splitter allowing dual column, dual FID analysis. NEW BAC UI columns (123-9334UI-INT and 123-9434UI- INT) were used which improve resolution of critical analytes
7697 Headspace parameters 7697 Headspace Sampler Set point Oven 70 C Loop 70 C Transfer line 90 C Vial equilibration time Injection duration Vial size Vial shaking Vial fill mode Vial fill pressure Loop ramp rate Loop final pressure Loop equilibration time 7 min 0.5 min 20 ml Off Default (50 ml/min to 15 psi (0.1 min)) 15 psi 30 psi/min 1.5 psi 0.05 min
Intuvo Parameters Intuvo 9000 GC Oven Set point 40 C (6.5 min) Split/Splitless Inlet Split 10:1, 110 C DB-BAC1 UI (123-9334UI-INT) 30m x 320µm x 1.8µm DB-BAC2 UI (1230-9434UI-INT) 30m x 320µm x 1.2µm Constant pressure 21 psi Controlled by column 1 FID (Front and Back) 250 C H2 Air 30 ml/min 400 ml/min N2 (makeup) 25 ml/min Jumper chip 110 C Bus Default (On 200 C) Front/Back Signal 20 Hz Jumper chip set to the inlet temperature Bus temperature set to default
Response Response Calibration Results Calibration standards were made in house 0.8% to 0.01% methanol, acetaldehyde (not quantified), acetone, ethanol, and isopropanol. Headspace vials were made in triplicate at each level consisting of 450 µl of internal standard (0.3% n- propanol) and 50µL of standard. Calibration curves for methanol, ethanol, acetone, and isopropanol are shown right. Ethanol calibration curves yield 0.9996 or better for the dual column ensemble. Slope difference between the two column/detector pairs is 6.3% 1000 900 800 700 600 500 400 300 200 100 0 1000 900 800 700 600 500 400 300 200 100 0 BAC UI 1 y = 2.0265x + 4.254 R² = 0.999 y = 1.1191x + 2.5958 R² = 0.9887 y = 0.3281x + 0.9655 R² = 0.9973 0 100 200 300 400 500 600 700 800 900 Concentraion (mg/dl) BAC UI 2 y = 2.1435x + 1.2863 R² = 0.9991 y = 1.2208x + 2.8953 R² = 0.9868 y = 0.3549x + 0.5807 R² = 0.9983 0 100 200 300 400 500 600 700 800 900 Concentraion (mg/dl) y = 0.616x - 1.3505 R² = 0.9996 Isopropanol Acetone Ethanol Methanol y = 0.6554x - 1.1943 R² = 0.9997 Isopropanol Acetone Ethanol Methanol
Calibration Accuracy Verification Ethanol Standard Calculated Concentration DB-BAC1 UI Pass/Fail Calculated Concentration DB-BAC2 UI Pass/Fail Agilent Ethanol Standards were evaluated on the Intuvo 9000 GC. The concentration of the ethanol standards was calculated based on the calibration curves and compared to the expected concentration. Pass or fail was determined with ± 6% error tolerance. 20 mg/dl (5190-9756) 50 mg/dl (5190-9757) 80 mg/dl (5190-9758) 100 mg/dl (5190-9759) 150 mg/dl (5190-9760) 200 mg/dl (5190-9761) 300 mg/dl (5190-9762) 400 mg/dl (5190-9763) 19.8 mg/dl Pass 19.3 mg/dl Pass 50.0 mg/dl Pass 47.1 mg/dl Pass 79.3 mg/dl Pass 76.8 mg/dl Pass 96.7 mg/dl Pass 94.4 mg/dl Pass 152 mg/dl Pass 149 mg/dl Pass 197 mg/dl Pass 193 mg/dl Pass 302 mg/dl Pass 302 mg/dl Pass 384 mg/dl Pass 386 mg/dl Pass
Area Repeatability Area repeatability for the 80mg/dL standard as well as the Agilent Blood Alcohol Checkout mix (5190-9765) was determined for five replicate headspace vials. Analyte DB-BAC1 UI DB-BAC2 UI Ethanol 80 mg/dl standard 3.70% 2.80% Methanol 4.10% 1.40% Acetaldehyde 2.80% 3.00% Ethanol 2.30% 1.10% Isopropanol 3.30% 1.90% t-butanol 2.80% 2.70% Propanal 3.40% 3.00% 4.1% or better! n-propanol 3.10% 2.10% Acetone 3.40% 2.90% Acetonitrile 2.30% 2.80% 2-Butanol 2.00% 3.00% Ethyl acetate 3.20% 3.10% 2-Butanone 3.10% 3.00%
Retention Time Repeatability Retention time repeatability for the 80mg/dL standard and the Agilent Blood Alcohol Checkout mix (5190-9765) was determined for five replicate headspace vials. Analyte DB-BAC1 UI DB-BAC2 UI Ethanol 80 mg/dl standard 0.04% 0.10% Methanol 0.01% 0.02% Acetaldehyde 0.01% 0.02% Ethanol 0.02% 0.05% Isopropanol 0.02% 0.04% t-butanol 0.03% 0.04% Propanal 0.01% 0.02% 0.1% or better! n-propanol 0.03% 0.04% Acetone 0.02% 0.03% Acetonitrile 0.02% 0.03% 2-Butanol 0.04% 0.04% Ethyl acetate 0.02% 0.03% 2-Butanone 0.02% 0.03%
DB-BAC1 UI Blood Alcohol Checkout Mix New columns resolve t-butanol and n-propanol from analytes of interest 2 Average resolution = 2.6 6 7 1. Methanol 2. Acetaldehyde 3. Ethanol 4. Isopropanol 5. t-butanol 6. Propanal 7. n-propanol 8. Acetone 9. Acetonitrile 10.2-Butanol 11.Ethyl Acetate 12.2-Butanone 5 8 11 12 1 3 4 9 10
DB-BAC2 UI Blood Alcohol Checkout Mix Elution order changes allow confirmation of analyte identification 2 6 8 5 7 12 11 1. Methanol 2. Acetaldehyde 3. Ethanol 4. Isopropanol 5. t-butanol 6. Propanal 7. n-propanol 8. Acetone 9. Acetonitrile 10.2-Butanol 11.Ethyl Acetate 12.2-Butanone 1 3 9 4 10
Conclusions Dual column analyses can be easily accomplished with Intuvo Analysis of USP <467> and blood alcohol can be easily accomplished with Intuvo Inlet splitting capability enables analysis and confirmation in a single run on two columns of different phases Can be configured as an Agilent Analyzer as well Includes a factory method and report template New columns (DB-624 Select UI, DB-Wax UI, DB-BAC 1 UI and DB-BAC 2 UI) yield excellent resolution of the analytes of interest in their application space. Excellent linearity, retention time repeatability, and area repeatability were achieved with both applications configured with the inlet splitter.
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