06-SIP-GC-044-EN Extractable Study of Container Closure Pack Used to Store Oral Drug Product Using Dynamic Headspace Technique : SHIMADZU (Shimadzu Corporation)

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User Benefits Application News Extractable Study of Container Closure Pack Used to Store Oral Drug Product Using Dynamic Headspace Technique  ASSP and simultaneous Scan/MRM analysis facilitating accurate qualitative identification and quantitation at the same time.  Dynamic headspace enables concentrating the analytes in cold trap with multi-injection count and helps to achieve higher sensitivity over static headspace, especially for trace level quantitation. Hemant Kesarkar, Prashant Hase, Durvesh Sawant, Amol Ramishte, Siddhesh Sabadra, Dheeraj Handique, Pratap Rasam and Jitendra Kelkar Shimadzu India Pvt. Ltd. Gas Chromatograph Mass Spectrometer GCMS-TQ 8050 NX, HS-20 NX Trap  Introduction Overview: Extractable and leachable (E & L) studies are becoming increasingly important in the pharmaceutical industry as it is a mandatory requirement from FDA during filing of the drug product. The purpose of E & L studies is to identify and evaluate possible toxicological risks. In this study, the regulatory references aim to identify traces of potential chemical substances. These substances may be harmful to patients due to their toxicity or may impact the activity of the drug product. Hence, to ensure the safety and efficacy of the drug throughout its shelf-life, E & L studies play an important role. What Are Extractables and Leachables ? Extractables are organic and inorganic chemical entities that are released from a pharmaceutical packaging/delivery system, packaging component, or packaging material of construction into an extraction solvent under laboratory conditions [1] . Leachables are foreign organic and inorganic chemical entities that are present in a packaged drug product because they have leached into the packaged drug product from a packaging/delivery system, packaging component, or packaging material of construction under normal conditions of storage and use or during accelerated drug product stability studies [1] . Extractables Drug Degradation product Leachables Figure 1: Extractables, Leachables and Drug degradation product Safety Thresholds: Product Quality Research Institute (PQRI) has specified the control thresholds like Safety Concern Threshold (SCT) and Analytical Evaluation Threshold (AET) to guide the direction for initial assessment. SCT is the threshold below which a leachable has a dose so low that it presents negligible carcinogenic and non-carcinogenic toxic effects. In PQRI, it is specified as 1.5 µg/day for Parenteral and Ophthalmic Drug Products (PODP). AET is the threshold at or above which a leachable should be characterized and reported for toxicological assessment. The AET can be mathematically derived from the SCT based on the factor that includes the dosing parameters of the drug product. For calculating the AET, formulae reported in the PQRI recommendation document were followed [2] . Sources of Extractables: Extractables are derived from a variety of sources and exhibit extensive chemical diversity. Few of the primary sources are listed below. i. Chemical additives in individual polymeric packaging material ii. Chemical entities that are present in the packaging components iii. Monomers and higher molecular mass oligomers derived from incomplete polymerization iv. Migrants from secondary and tertiary packaging materials such as inks, label adhesives etc. v. Surface residues on metal canisters and containers vi. Chemical substances on the surface of components fabrication machinery such as anti-static agents. Before designing the extractable or leachable study, calculation of AET is a must. To calculate the AET, information of drug product such as maximum daily dose (MDD), Pack size, number of dose per container closure pack (CCP), root of administration and respective SCT etc is required. Penicillin V was available in lab and the same was considered for AET calculation. Some of the parameters which were considered for this calculation are mentioned in Table 1. Table 1: Product information for AET calculation Figure 1 depicts hypothetical relation between Extractables, Leachables and Drug Degradation products. Parameter Value Name of the product Penicillin V Route of administration Oral Pack size (mL) 100 mL MDD (mL/day) 20 mL considering 4 doses of 5 mL Material of construction (MOC) of CCP Plastic, Glass SCT for OINDP 1.5 µg/day Posology or strength 125 mg / 5 mL and 250 mg / mL Calculation of AET: SCT (µg/Day) AET = --------------------- × No. of doses per CCP (µg/Container) MDD (mL/Day) 1.5 (µg/Day) = ------------------- × 20 doses /pack 4 (Doses/Day) = 7.5 (µg/Pack) After applying uncertainty of 0.5 : 3.75 (µg/pack) For liquid dosage form = 3.75 (µg/pack) / Pack size (mL) =3.75 µg / 100 mL = 0.0375 ppm

Application News Figure 2: Typical images of CCP  Experimental Design There are many ways by which extractable study can be designed like generating extract by maceration (solvent soaking), by reflux/Soxhlet/sealed vessel/sonication or solvent extraction (manually and automated) etc [1] . However, one can perform this study by directly heating the CCP using the headspace injection technique. Physical and chemical properties of the drug product such as pH, polarity and chemical composition plays an important role and need to be considered while designing the extraction experiments. As per above calculation, if the CCP under study is supposed to be used for the storage of the Penicillin V with above dosage form, then it should not leach any chemical above 0.0375 ppm. And if any chemical entity observed above this threshold, then identification and toxicological assessment of that entity is required. Figure 2 depicts typical image of CCP under study. Note: Uncertainty factor is generally applied to overcome the risk associated due to extraction error and to evaluate the toxic entities below its accepted level. Experiment-A (Incubation with acidic media): To mimic the condition, acidic media was selected for evaluation. Two bottle caps were incubated at 85 ˚C for 72 hours with 50 mL Formic acid media having pH of 2. After incubation, 25 mL of incubated solution inside the packaging material was extracted using 25 mL of Ethyl acetate (EA) and the layer was transferred to a test tube prefilled with Sodium sulfate. Experiment-B-1 (Reflux with Polar solvent): Product under study contains water as a major constituent, which is highly polar. To mimic the same, polar solvent was considered for reflux experiment. One cap of the CCP was cut in the small pieces and refluxed with 50 mL Ethanol at 80 ˚C for 2 hrs. Then this refluxed solution was cooled at room temperature, transferred 10 mL of solution from reflux flask in a test tube and evaporated till dryness using nitrogen evaporator. Contents left in the tube were re-constituted with 1 mL of Methanol, sonicated, filtered through 0.2 µ syringe filter and used for analysis. Experiment-B-2 (Reflux with Non-polar solvent): This experiment was performed in the same way as stated in the B-1. Only Hexane was used as reflux medium instead of Ethanol. Non-polar solvent was selected for reflux considering worst case scenario. Entire experimental design flowchart is depicted in Figure 3 below. Extractable study is performed at accelerated temperature conditions considering worst case scenario for storage of the drug product. Since measured pH for the final drug product inside was slightly acidic, we conducted extractable study at similar pH by refluxing the CCP with solvents of different polarities. This scenario will be more effective in focusing on extractables that can migrate into the drug product, thereby increasing the probability of detecting them as potential leachables. As a result, it allows for a more realistic assessment of potential risks associated with the packaging or material components under actual use conditions. Analysis plan Extract by incubation Extract by reflux 2 Bottle caps  incubation with 50 mL acidic media of formic acid (pH=2.0) at 85 ˚C for 72 hrs 25 mL of solution + 25 mL EA 25 mL EA layer dried with Na 2 SO 4  concentrated to 2 mL Analyzed by HS-Trap technique with GCMS Quantitation of extractables Reflux with polar solvent 1 bottle cap refluxed with 50 mL Ethanol at 80 ˚C for 2 hr 10 mL of reflux concentrated to 1 mL  Headspace-GCMS Quantitation of extractables Reflux with non-polar solvent 1 bottle cap refluxed with 50 mL Hexane at 80 ˚C for 2 hr 10 mL of reflux concentrated to 1 mL Headspace-GCMS Quantitation of extractables Content of known extractables are reported by quantification from MRM data. Unknown peaks were identified with library search and their contents were reported by semi-quantitation method Figure 3 : Detailed layout of the experiments designed for sample preparation

Application News 0.0 5.0 10.0 15.0 20.0 Conc. 0 5000000 10000000 15000000 20000000 Area In this method, fifteen standards were analyzed by GCMS- TQ8050 NX with HS-20 NX (Trap) autosampler. For the method suitability, several important parameters like system suitability, linearity and precision were performed. Instrument parameters and MRM transitions are shown in Tables 2 and 3, respectively.  Methods of Analysis MRM Transitions Compound name Target MRM CE-1 Reference MRM CE-2 Toluene D8 100.00>98.10 19 98.00>70.10 19 Cyclotrisiloxane, hexamethyl- 207.00>191.00 17 207.00>119.10 27 Alpha methyl benzene 117.00>115.10 13 117.00>91.10 19 2-Octanone 58.00>43.00 9 59.00>43.10 27 Isobutyl benzene 91.00>65.10 19 134.00>91.10 19 Cyclopentasiloxane, decamethyl- 73.00>45.00 13 267.00>250.90 19 Tridecane 71.00>43.10 9 85.00>43.10 9 1,1'-Biphenyl, 2-fluoro- 172.00>170.10 25 172.00>151.10 25 Tetradecane 71.00>43.10 9 85.00>43.10 9 Butyrated Hydroxy Toluene 205.00>57.10 19 220.00>205.20 13 2,6-Di-tert-butyl-4- ethylphenol 219.00>57.10 19 234.00>219.20 13 Benzophenone 105.00>77.10 13 105.00>95.10 19 3,5-di-tert-Butyl-4- hydroxybenzaldehyde 219.00>191.20 9 219.00>175.10 17 Eicosane 71.00>43.10 9 85.00>43.10 9 Docosane 71.00>43.10 9 85.00>43.10 9 Instrument used : GCMS-TQ8050 NX with HS-20 NX (Trap) Column : SH-I-5Sil MS 0.25 mm I.D. X 30 m d.f=0.25 µm (P/N: 221-75954-30) Injection Mode : Split (P/N : 227-35007-01) Split ratio : 10 Flow control mode : Linear velocity Linear velocity : 36 cm/sec Carrier gas : Helium Temp. Program : 40 ˚C (1 min), 7 ˚C/min to 110 ˚C (3 min), 10 ˚C/min to 280 ˚C (4 min) Ionization Mode : Electron Impact Ion source Temp. : 220 ˚C Interface Temp. : 280 ˚C Acq. Mode : SCAN/MRM Scan Range : 45–500 m/z Detector Voltage : Optimized by adjusting the intensity of m/z 314 = 500000 in autotuning Standard chemicals which are listed above, were procured and appropriately diluted with Methanol and Water to achieve the desired concentration as mentioned in Table 4. Preparation steps were followed as shown in Figure 4. Linearity of the all above listed compounds was performed in the SCAN/MRM mode and used for semi-quantitation of unknown extractables. Parameters for Dynamic Headspace: Mode : Trap Oven Temp. : 120 ˚C Sample Line Temp. : 170 ˚C Transfer Line Temp. : 200 ˚C Trap cooling Temp. : -20 ˚C Trap Desorb. Temp. : 280 ˚C Trap Equilibrium Temp. : -20 ˚C Shaking Level : 5 Multi Injection Count : 3 Pressurization Gas Pressure : 130 kPa Dry Purge Gas Pressure : 40 kPa Equilibrating Time : 10 min Dry Purge Time : 0 min Injection Time : 25 min Needle Flush Time : 25 min GC Cycle Time : 45 min • Prepared 10 ppm mix standard stock of all targeted impurities in Methanol • From this stock, prepared linear level standards such as 0.1 ppm, 0.3 ppm, 0.5 ppm, 1 ppm and 2 ppm in methanol • Transferred 1 mL MS grade water in a 20 mL HS vial, added 100 mg sodium sulfate and 25 µL of linearity level standard • Crimped carefully and submitted for GCMS analysis Figure 5 to 10 depicts data for some of the representative analytes. Each figure shows data for one representative analyte and divided into 4 sections as follows, (a) calibration curve and (b) chromatogram at LOQ level for MRM mode. Similarly, (c) and (d) shows data for TIC response in SCAN mode . Toluene D8 Figure 5: Data for Toluene D8 Hexamethyl Cyclotrisiloxane c d 0.0 5.0 10.0 15.0 20.0 Conc. 0 1000000 2000000 3000000 4000000 Area 3.5 4.0 4.5 5.0 0 25000 50000 75000 2:98.00>70.10 2:100.00>98.10 a b 3.5 4.0 4.5 250000 500000 750000 1000000 1250000 1500000 1:TIC Figure 6: Data for Hexamethyl Cyclotrisiloxane c d a b 0 10 20 Conc. 0 1000000 2000000 3000000 4000000 Area 4.0 4.5 5.0 5.5 0 25000 50000 75000 100000 125000 3:207.00>119.10 3:207.00>191.00 0 10 20 Conc. 0 2500000 5000000 7500000 10000000 12500000 Area 4.0 4.5 5.0 5.5 250000 500000 750000 1000000 1250000 1500000 1750000 1:TIC  Standard Preparation  Results and Discussion Table 2: Instrument parameters Table 3: MRM transitions Figure 4 : Standard preparation procedure

Application News 0.0 25.0 Conc. 0 2500000 5000000 7500000 Area 8.0 8.5 9.0 9.5 0 500000 1000000 1500000 2000000 2500000 3000000 1:TIC 8.0 8.5 9.0 9.5 0 25000 50000 75000 4:134.00>91.10 4:91.00>65.10 7.5 8.0 8.5 9.0 250000 500000 750000 1000000 1250000 1500000 1750000 1:TIC 7.5 8.0 8.5 9.0 0 10000 20000 30000 40000 2:117.00>91.10 2:117.00>115.10 Figure 7: Data for Alpha Methyl Styrene c d a b Alpha Methyl Styrene 0.0 25.0 Conc. 0 2500000 5000000 Area 0.0 25.0 Conc. 0 10000000 20000000 30000000 40000000 50000000 Area Figure 9: Isobutyl Benzene c d a b Isobutyl Benzene 0.0 25.0 Conc. 0 2500000 5000000 Area 0 10 20 Conc. 0 10000000 20000000 30000000 Area Decamethyl Cyclopentasiloxane 10.5 11.0 11.5 12.0 250000 500000 750000 1000000 1250000 1500000 1750000 1:TIC 10.5 11.0 11.5 12.0 0 50000 100000 150000 200000 250000 2:267.00>250.90 2:73.00>45.00 Figure 8: Decamethyl Cyclopentasiloxane c d a b 0.0 25.0 Conc. 0 25000000 50000000 75000000 Area Figure 10: 2,6-Di-tert-butyl-4-ethylphenol c d a b 2,6-Di-tert-butyl-4-ethylphenol 0.0 25.0 Conc. 0 1000000 2000000 3000000 Area 20.0 20.5 21.0 21.5 0 10000 20000 30000 40000 50000 3:234.00>219.20 3:219.00>57.10 0.0 25.0 Conc. 0 10000000 20000000 30000000 40000000 50000000 Area 20.0 20.5 21.0 21.5 500000 1000000 1500000 2000000 2500000 1:TIC Sr. No. Compound Name RT (min) Linearity range (ppb) LOQ (ppb) Correlation coefficient (r 2 ) S/N* ratio at LOQ MRM SCAN 1 Toluene D8 4.0 2.5 to 25 2.5 1.000 0.998 1530 2 Cyclotrisiloxane, hexamethyl- 4.7 2.5 to 25 2.5 0.997 0.994 93 3 Alpha methyl benzene 8.1 2.5 to 50 2.5 1.000 0.999 70 4 2-Octanone 8.2 2.5 to 50 2.5 0.995 0.992 32 5 Isobutyl benzene 8.6 2.5 to 25 2.5 0.999 0.993 71 6 Cyclopentasiloxane, decamethyl- 11.3 2.5 to 50 2.5 0.998 0.994 66 7 Tridecane 16.1 2.5 to 50 2.5 0.997 0.994 543 8 1,1'-Biphenyl, 2-fluoro- 17.6 2.5 to 50 2.5 0.997 0.991 11 9 Tetradecane 18.2 2.5 to 50 2.5 0.991 0.991 47 10 Butyrated Hydroxy Toluene 20.0 2.5 to 50 2.5 0.997 0.991 11 11 2,6-Di-tert-butyl-4-ethylphenol 20.7 2.5 to 50 2.5 0.999 1.000 31 12 Benzophenone 21.9 2.5 to 50 2.5 0.997 0.991 1589 13 3,5-di-tert-Butyl-4-hydroxybenzaldehyde 23.4 2.5 to 50 2.5 0.995 0.994 11 14 Eicosane 25.8 2.5 to 50 2.5 0.991 0.991 2676 15 Docosane 27.7 2.5 to 50 2.5 0.998 0.996 11 Table 4 : Results of standard acquisition *Note: Reported S/N values are from MRM acquisition and calculated as per ASTM method. Reported concentration are as such concentrations. Fifteen compound were selected for analysis which generally observed in extractable study and they have potential to leach into the drug product. Results obtained from the standard data acquisition are reported as shown in the below Table 4. All the compounds are complying with the acceptance criteria for correlation coefficient (not less than 0.99) and S/N ratio (not less than 10). The noise levels in SCAN mode are generally high. This can be further confirmed by the LOQ chromatograms displayed in section (d) of each figure. However, this data were used for semi-quantitation of unknown extractable impurities only. Average of slope and intercept values from the calibration plot of targeted compounds were used for the quantifying unknown impurities.

Application News The commonly available technique across all the instrument provider is static headspace injection technique, wherein only limited volume of the analyte gas will be used for the analysis. Generally, it is 1 mL and not sufficient to achieve the sensitivity at trace level. Shimadzu’s dynamic headspace technique enables multiple headspace extractions (up to 10 times) from the same vial and allows to concentrate the analytes on the cold trap. This enhances the sensitivity and allows trace level detection which static headspace fails to achieve under similar conditions. Furthermore, users can perform maximum headspace extractions from same vial up to 10 times and enhance the method capability to detect the analytes at trace level where static headspace technique fails to achieve under similar condition. Figure 11 depicts Shimadzu GCMS-TQ8050 NX instrument with HS-20 NX (Trap) autosampler. VOC screening Direct sample analysis in headspace 0.5 g of cap sample in a 20 mL HS vial (P/N : 227-34140-01) Analyzed in static and dynamic HS Identified potential extractable and reported for information purpose Figure 12: Sample preparation procedure and image of the HS vial with sample Figure 13: Overlayed chromatograms of sample analyzed by static mode and dynamic mode with blank injection In the sample analyzed by static headspace (with 1 mL loop), only few peaks were detected. When the same sample was injected in dynamic headspace keeping other method parameters same, the number of compounds detected increased drastically. The chromatograms of CCP sample obtained in both the techniques and air blank are compared in Figure 13. (c) 0.5 g cap sample in trap mode HS (b) 0.5 g cap sample in loop mode HS (a) Air blank injection 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 (x10,000,000) (c) (b) (a) Extracts were prepared by two techniques as stated under experimental design. From which, Figure 14 depicts the chromatogram of the sample extract which was prepared by incubation of the sample in acidic media for 72 hours and at 85 °C. 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min 0 250000 500000 750000 1000000 1250000 1500000 1750000 2000000 2250000 2500000 2750000 3000000 3250000 3500000 3750000 Figure 14: Chromatogram of sample incubated with acidic media for 72 hours at 85 °C Figure 11: Shimadzu GCMS-TQ8050 NX with HS-20 NX (Trap) In the incubated extract, only few compounds were detected which were semi-quantified using standard data. Content for all these peaks were found to be below AET level. Table 5 elaborates the results of incubation experiment. To compare the sensitivities between these two-injection techniques especially for extractable and leachable study, we performed the screening of this CCP. The steps are shown in Figure 12.

Application News 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min 0 2500000 5000000 7500000 10000000 12500000 15000000 17500000 20000000 22500000 25000000 27500000 30000000 32500000 35000000 37500000 40000000 42500000 Figure 16: Chromatogram of sample refluxed with Ethanol for 2 hours at 80 °C RT (min) Identified name of the unknown imp. SI Content (PPM) 3.75 1,3,5-Cycloheptatriene 87 0.00 3.83 Acetic acid, butyl ester 85 0.00 4.44 Cyclotrisiloxane, hexamethyl- 74 0.01 7.85 Cyclotetrasiloxane, octamethyl- 67 0.00 9.51 Octane, 3-ethyl-2,7-dimethyl- 87 0.00 15.31 Decane, 2,3,5,8-tetramethyl- 84 0.00 18.16 Octane, 3,4,5,6-tetramethyl- 78 0.00 19.22 Decyl isopropyl ether 50 0.00 19.61 Heptacosane 87 0.00 19.99 2,4-Di-tert-butylphenol 89 0.01 23.93 Isopropyl myristate 79 0.00 24.38 Phthalic acid, isobutyl nonyl ester 83 0.00 28.64 Cyclodecasiloxane, eicosamethyl- 56 0.00 30.71 Cyclononasiloxane, octadecamethyl- 67 0.01 31.70 Cyclooctasiloxane, hexadecamethyl- 55 0.00 Table 5: Results of incubation experiment Also, same CCP was analyzed by preparing extract by reflux experiment which is performed considering its importance in leachable assessment and investigating the source of any unknown impurity detected in the future sample. Here, two solvents were taken for reflux i.e Ethanol to extract polar organic impurities and Hexane to extract non-polar organic impurities. Samples were refluxed as stated in the Figure 15 and analyzed by GCMS technique. Figure 16 depicts the chromatogram of sample refluxed with Ethanol and Figure 17 depicts the chromatogram of sample refluxed with Hexane. There were more number of peaks in the Hexane reflux sample in comparison with Ethanol reflux sample. All the peaks were integrated and their probable names were reported as per NIST- 23 library. Also, their semi-quantitated content is reported below in Table 6. Only content from Hexane reflux sample is reported considering the worst-case scenario. • Cap sample was cut into small pieces and transferred into reflux flask • Added 50 mL Reflux solvent (Ethanol / Hexane) + pieces of porcelain • Refluxed at boiling point for 2 hr • Solution was transferred to 100 mL volumetric flask and make up to 100 mL by Hexane • 10 mL of this solution was evaporated in the Nitrogen evaporator • Reconstituted with 1 mL of methanol • Used for analysis in GCMS Figure 15: Procedure for obtaining sample extract by reflux method Figure 17: Chromatogram of sample refluxed with Hexane for 2 hours at 70 °C 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min 0 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 5000000 5500000

Application News Here, most of the extractables show their presence above AET level in the CCP under study. It means that those chemical compounds were present in the CCP and leached out when exposed to the Hexane under hot condition. Hexane reflux condition is not a reasonable with respect to the nature and storage condition of the drug product inside. On the other hand, this CCP will be used to store the Penicillin V product which is supposed to be consumed with water and pH of the final solution is slightly acidic. Hence, incubation with acidic- aqueous medium will be more relatable for this CCP considering real-time scenario. However, this data can be considered for leachable assessment, wherein user must identify the source of the particular leachable. Table 6 : Results of Unknown compounds Sr. No. Name of Unknown extractable RT (min) SI Content (ppm) 1 Dodecane, 2,6,11-trimethyl- 13.13 92 0.44 2 Decane, 2,3,5,8-tetramethyl- 15.43 93 0.51 3 Decane, 3,7-dimethyl- 16.59 94 1.18 4 Dodecane, 4,6-dimethyl- 16.81 92 0.65 5 Hexadecane 18.23 95 1.09 6 Tridecane, 4-methyl- 19.31 87 0.95 7 Decane, 2,3,5,8-tetramethyl- 19.47 90 0.5 8 Heptadecane, 2,6,10,15-tetramethyl- 19.62 91 0.82 9 Heptadecane, 2,6,10,15-tetramethyl- 19.69 93 3.48 10 Dodecane, 2,6,11-trimethyl- 19.84 92 1.01 11 2,4-Di-tert-butylphenol 20.07 93 5.95 12 11-Methyldodecanol 20.12 90 1.87 13 11-Methyldodecanol 20.27 88 1.06 14 Dodecane, 1-iodo- 20.32 91 1.19 15 Dodecane, 2,6,11-trimethyl- 20.38 93 7.54 16 Nonane, 3-methyl-5-propyl- 20.42 88 0.88 17 Carbonic acid, eicosyl vinyl ester 20.53 92 3.77 18 Nonadecane 20.67 93 4.11 19 Heptacosane 20.72 85 1.05 20 Heptadecane, 2,6,10,15-tetramethyl- 20.83 94 2.6 21 Heptadecane, 2,6,10,15-tetramethyl- 20.93 94 0.95 22 Heptadecane, 2,6,10,15-tetramethyl- 20.98 93 1.04 23 Dodecane, 4,6-dimethyl- 21.08 92 0.53 24 Pentanoic acid, 2,2,4-trimethyl-3- carboxyisopropyl, isobutyl ester 21.18 88 0.64 25 Hexadecane 21.33 94 0.92 26 Heneicosane 22.57 93 4.86 27 Hexadecane 22.68 93 1.29 28 11-Methyldodecanol 22.92 89 2.01 29 11-Methyldodecanol 23.03 88 1.28 30 Heneicosane 23.09 93 9.35 31 Carbonic acid, eicosyl vinyl ester 23.13 88 2.2 32 Heneicosane 23.2 93 3.96 33 Cyclohexane, 1-ethyl-2-propyl- 23.25 82 2.1 34 3-Ethyl-2,6,10-trimethylundecane 23.31 90 3.63 35 1,7-Dimethyl-4-(1- methylethyl)cyclodecane 23.35 85 2.52 36 Hexadecane 23.42 93 2.35 37 Carbonic acid, eicosyl vinyl ester 23.48 90 1.82 38 Decane, 3,7-dimethyl- 23.53 84 0.57 39 Hexadecane 23.57 92 1.5 40 1-Dodecanol, 2-hexyl- 23.62 88 1.4 41 Carbonic acid, eicosyl vinyl ester 23.68 92 0.97 42 Dodecane, 1-iodo- 23.8 87 0.69 43 Carbonic acid, hexadecyl prop-1-en-2-yl ester 23.84 90 1.63 44 Isopropyl myristate 24.01 87 8.88 45 Dodecane, 4,6-dimethyl- 24.74 89 0.63 46 Heneicosane 24.92 93 5.08 47 3-Ethyl-3-methylheptane 24.96 79 1.01 48 Tridecanol, 2-ethyl-2-methyl- 25 79 2.14 49 Hexadecanoic acid, methyl ester 25.1 86 1.94 50 1-Decanol, 2-hexyl- 25.23 91 2.78 51 Tetracontane 25.3 86 1.86 52 Eicosane 25.36 93 13.19 53 Octacosane, 1-iodo- 25.4 85 2.85 54 Heptadecane 25.44 92 4.81 55 Heneicosane 25.53 92 7.01 56 Hexatriacontyl trifluoroacetate 25.59 77 1.61 57 Heneicosane 25.62 91 4.09 58 Eicosane 25.74 90 5.08 59 Eicosane 25.83 85 4.91 60 Carbonic acid, octadecyl vinyl ester 25.96 89 2.74 Sr. No. Name of Unknown extractable RT (min) SI Content (ppm) 61 1-Naphthalenepropanol, .alpha.- ethenyldecahydro-2-hydroxy- .alpha.,2,5,5,8a-pentamethyl-, [1R- [1.alpha.(R*),2.beta.,4a.beta.,8a 26.03 71 0.65 62 1-Decanol, 2-hexyl- 26.08 84 0.72 63 11-Methyltricosane 26.24 81 1.13 64 Cyclohexene, 3-(1-methylethyl)- 26.6 77 1.06 65 Heptafluorobutyric acid, pentadecyl ester 26.69 81 0.67 66 Dodecane, 2,6,11-trimethyl- 26.8 90 0.56 67 Eicosane 26.97 91 5.08 68 Tridecanoic acid, 4,8,12-trimethyl-, methyl ester 27.05 80 4.13 69 1-Decanol, 2-hexyl- 27.26 89 2.98 70 Eicosane 27.36 92 12.63 71 Heptadecane 27.43 89 5.98 72 Eicosane 27.51 90 5.83 73 Eicosane 27.58 92 4.33 74 Carbonic acid, decyl nonyl ester 27.62 79 2.45 75 Dotriacontane, 1-iodo- 27.69 82 3.86 76 Tetratriacontyl heptafluorobutyrate 27.71 83 3.06 77 Eicosane 27.76 88 2.52 78 1-Decanol, 2-hexyl- 27.82 85 1.9 79 Tetrapentacontane 27.88 77 1.3 80 11-Methyldodecanol 27.92 85 2.56 81 11-Methyltricosane 27.99 88 1.37 82 Eicosane 28.06 87 0.72 83 Nonahexacontanoic acid 28.13 85 0.65 84 1-Decanol, 2-hexyl- 28.24 79 0.98 85 Hexacosane 28.58 95 2.17 86 Eicosane 28.82 91 2.89 87 Tetrapentacontane 28.89 88 1.28 88 Pentatriacontane 29.1 83 1.83 89 Eicosane 29.17 92 7.36 90 Eicosane 29.24 92 2.85 91 Eicosane 29.31 89 2.46 92 Tetrapentacontane 29.37 87 2.25 93 Heneicosane 29.42 93 7.03 94 Eicosane 29.52 84 1.74 95 Tetrapentacontane, 1,54-dibromo- 29.67 85 1.38 96 Octacosane, 2-methyl- 30.23 94 2.84 97 Tetrapentacontane 30.52 89 1.07 98 Tetrapentacontane 30.57 80 0.7 99 Tetrapentacontane 30.83 89 2.4 100 Triacontane, 1-iodo- 30.89 86 2 101 Tetratetracontane 31 94 3.3 102 Tetratetracontane 31.8 91 0.75 Continued…..

Application News www.shimadzu.com/an/ Shimadzu Corporation Copyright © 2026 Shimadzu Corporation and/or its affiliates. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. See http://www.shimadzu.com/about/trademarks/index.html for details. Third party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not they are used with trademark symbol “TM” or “”. Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Shimadzu India Pvt. Ltd. www.shimadzu.in First Edition: Jun. 2026 06-SIP-GC-044-EN  Conclusion  The chromatographic profile of solvent reflux experiment shows higher number of extractables than aqueous incubation. Furthermore, in aqueous incubation, the content of extractables is well below the AET level. Since, placebo of the ophthalmic drug product is completely aqueous, CCP used in the analysis is totally safe and can be used for the storage of Penicillin V.  Shimadzu’s GCMS-TQ provides high scan speed of 20000 amu/sec with ASSP technology enabling simultaneous SCAN/MRM analysis which is crucial in E&L study.  Generally, if the drug product falls under parenteral and oral drug product (PODP), evaluation threshold will be at higher side due to having wider SCT. However, AET for Penicillin V was at too low to evaluate with liquid injection and static headspace technique. This has been proved with the trap headspace technology.  To analyze the complete extractable profile with the sample screening is only possible with dynamic headspace as most of the extractables were not detected in the injection acquired with static headspace.  References [1] USP <1663> Assessment of Extractables Associated with Pharmaceutical Packaging/Delivery Systems [2] Product Quality Research Institute “Safety Thresholds and Best Demonstrated Practices for Extractables and Leachables in Parenteral Drug Products (Intravenous, Subcutaneous, and Intramuscular) ” https ://pqri.org/wp-content/uploads/2022/03/PQRI-PDP- Recommendation-2022.pdf GCMS-TQ and ASSP are trademarks of Shimadzu Corporation or its affiliated companies in Japan and/or other countries.

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