9 Summary


The results presented in this work demonstrate that the TRACE 1310 GC-FID fulfi ls the USP <467> requirements, meeting the suitability criteria for chromatographic separation as required for regulated c-GMP pharma laboratories. The equivalency of the method parameters assures a safe portability of the method from different HS-GC brands using the Valve-and-Loop headspace technology.


Separation of 37 fatty acid methyl esters according to AOAC method 996.06 by GC-FID Introduction


Food fat mainly consists of triglycerides and assessing the fat (trans and saturated) composition of food products as part of the nutritional information is a fundamental test for the food industry. The AOAC method 996.06 describes the determination of total, saturated and unsaturated fat in foods using capillary GC-FID by a multiple steps procedure: hydrolytic extraction followed by the derivatisation (methylation) of fatty acids to produce fatty acids methyl esters (FAMEs) which are the derivatives suitable for GC analysis [7].


Experimental


A TRACE 1610 Gas Chromatograph confi gured with an Instant Connect split/splitless SSL Injector and an Instant Connect Flame Ionisation Detector (FID) was coupled with an AI/ AS 1610 Autosampler and used to assess the chromatographic separation performance according to AOAC method 996.06.


A standard solution was prepared by diluting Restek Food Industry FAMEs mix (30 mg/mL in dichloromethane) (P/N 35077) to 1000 µg/mL in dichloromethane/hexane.


C23:3


quantitation and specifi c resolution requirements for critical peaks pair are included in AOAC method 996.06: (Rs


Results and discussion Chromatographic resolution (Rs


– C20:4 ). - C20:1


Table 1. Food Industry FAMEs mix (37 components) - identifi cation and retention times.


Peak


1 2 3 4


5 6


7


8 9


10 11 12 13 14 15 16


) is fundamental for FAMEs separation, identifi cation and ) ≥ 1.0 for FAMEs pair of adjacent peaks (C18:3


and C22:1 17 –


The chromatographic profi le of 37 FAMEs separation obtained with TRACE 1610 Gas Chromatograph (equipped with Restek Rt-2560 column) is shown in Figure 2; critical pair peaks are highlighted, and the achieved resolution meets and exceeds the requirements. Peak identifi cation and retention times are reported in Table 1.


18 19


C4:0 C6:0 C8:0


C10:0


C11:0 C12:0


C13:0 C14:0 C14:1 (c9) C15:0 Compound


Methyl butyrate Methyl caproate Methyl octanoate Methyl decanoate


Methyl undecanoate Methyl dodecanoate


Methyl tridecanoate Methyl myristate Methyl myristoleate Methyl pentadecanoate C15:1 (c10) Methyl C16:0 C16:1 (c9) C17:0


pentadecenoate Methyl palmitate


Methyl palmitoleate Methyl heptadecanoate C17:1 (c10) Methyl C18:0 C18:1 (t9) C18:1 (c9)


heptadecenoate Methyl stearate


Methyl


octadecenoate Methyl oleate


C18:2 (t9,t12) Methyl linolelaidate 41.779 42.333 43.284 43.695 44.776 34 35 36 37 C24:0 C20:5


(c5,c8,c11,c14,c17) C24:1 (c15)


C22:6 (c4,c7,c10,c13,c16,c19) 37.619 37.972 39.593 40.194 30 31 32 33 C20:3 (c11,c14,c17) C23:0 Methyl


eicosatrienoate Methyl


tricosanoate C20:4 (c5,c11,c14,c17) Methyl C22:2 (c13,c16)


arachidonate Methyl


docosadienoate Methyl lignocerate Methyl


eicosapentaenoate Methyl nervonate


Methyl docosahexaenoate 53.669 54.297 55.026 60.107 51.955 53.182 51.625 51.823


Retention Time (min)


11.937 14.101 17.770 22.704


25.375 28.050


30.667


33.201 35.235


35.634 Peak


20 21 22 23


24 25


26


27 28


29


C18:2 (c9,c12) C20:0


C18:3 (c6,c9,c12) C20:1 (c11)


C18:3 (c6,c9,c15) C21:0


C20:2 (c11,c14) C22:0 C20:3 (c8,c11,c14) C22:1 (c13) Compound


Methyl linoleate Methyl arachidate Methyl linolenate Methyl


eicosenoate


Methyl linolenate Methyl


heneicosanoate Methyl


eicosadienoate Methyl behenate Methyl


eicosatrienoate Methyl erucate


47.904 48.220


49.504


50.044 50.877


51.283


Retention Time (min)


45.679 46.341 47.125 47.622


Conclusion


The examples considered in this white paper demonstrate that the TRACE Series Gas Chromatograph systems allows for equivalent chromatographic performance ensuring that suitability requirements of specifi c regulatory methods are met. The application of method parameters within a standard working range for the gas chromatographic system, along with the use of standard consumables, allow for a smooth transfer maintaining the required analytical performance.


References


1. EMEA/CHMP/EWP/192217/2009 Rev. 1 Corr. 2** Committee for Medicinal Products for Human Use (CHMP).


2. FOOD AND DRUG ADMINISTRATION OFFICE OF REGULATORY AFFAIRS ORA Laboratory Manual Volume II, ORA-LAB.5.4.5 Methods, Method Verifi cation and Validation Revision #: 02 Revision June 2020.


3. USP Chapter 621 of the current United States Pharmacopeia.


4. Impurities: Guideline for Residual Solvents Q3C(R6), ICH Harmonised Guidelines, International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human use, 2016.


5. General Chapter USP <467> Organic Volatile impurities, Chemical Tests, United States Pharmacopeia, 2012 and Interim Revision Announcement Offi cial November 1, 2019; Offi cial December 1, 2020 <467> Residual Solvents.


6. Thermo Scientifi c White Paper 10705- Investigation of key parameters for a smooth method transfer to the new Thermo Scientifi c TriPlus 500 Headspace Autosampler


Figure 3. Chromatographic separation of a solvent standard of the Food Industry FAMEs mix (37 components) at 50 ppm (µg/mL) on column using the TRACE 1610 Gas Chromatograph and a Restek Rt-2560 column.


Summary


The TRACE 1610 Gas Chromatograph equipped with Restek RT-2560 100 m, 0.25 mm, 0.2 µm capillary column is suitable for FAMEs separation in food samples according to AOAC method 996.06, meeting or exceeding resolution requirements and providing reliable peaks integration and quantifi cation.


7. AOAC 996.06-1996 (2010) - Fat (Total, Saturated, and Unsaturated)


Please refer to WP-74062 for the full paper which will include: - Determination of gasoline range organics (GRO) in water by static headspace gas chromatography


- Separation of US EPA 16 priority polycylic aromatic hydrocarbons by GC-FID


For the full paper (WP-74062), please contact CMD.APACMarketing@thermofi sher.com


Safety Coated Glass Mobile Phase Reservoir Bottles


MicroSolv Technology’s™ collection of borosilicate glass, mobile phase bottles for HPLC, meet general laboratory safety standards. The exterior of these bottles is coated with a transparent, thick epoxy plastic. If dropped and broken, the plastic can prevent a spill incident, saving clean-up time and workers from being splashed with solvent or glass.


This special plastic coating is available on various bottle sizes including: a 1L, Standard 29/32 Taper Neck and bottles with a GL45 neck in 1L to 10L formats. Each GL45 neck bottle is supplied with a ‘drip ring’ and an inert closed screw cap. Compatible with many mobile phase fi ltration devices, you can safely fi lter directly into these bottles and cap them for storage or transportation.


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