Is Inert LC Column Hardware Beneficial for Pesticide or Mycotoxin Analysis?

For some years now, the development departments of high-pressure liquid chromatography companies have been dealing with the question of what influence the surfaces of the instrument and component hardware can have on peak shape and sensitivity in the detection of polar, chelating molecules. Biomolecules in particular, with their complex acid/base properties, but also compounds such as glyphosate and AMPA, with their organophosphate groups, show that the surface properties of the components in the sample path have a decisive influence on peak shape and sensitivity.

Recently, Restek developed a inert LC column line to address this issue more efficiently and effectively than short-term solutions, such as priming or medronic acid deactivation. Reports from several independent laboratories indicate that these new columns can offer substantial benefits for pesticides and mycotoxins analysis.

In June 2024, Maria Antonietta Carrera from the Department of Desertification and Geo-ecology, Experimental Station of Arid Zones in Almeria, under the supervision of Amadeo Fernandez-Alba from the European Union Reference Laboratory for Pesticide Residues in Fruit & Vegetables (EURL-FV), published a remarkable paper on Simultaneous analysis of pesticides and mycotoxins in primary processed foods: The case of bee pollen”, in which she compared a column commonly used for routine analysis of the analytes under consideration with a Raptor Inert ARC-18 column from Restek.

In her publication she describes “As reported in Fig. 3, no substantial differences were revealed by this test, since almost the same number of compounds could be detected at a concentration of 1 μg/kg with both columns. However, by looking at peaks obtained for about 15 % of analytes at 1 μg/kg, it was possible to notice a visible improvement in the peak shape (Fig. 4) when using the Raptor Inert column. This particular feature can be sometimes decisive for the identification of the analytes in real samples.”

Please find the full publication here: https://www.cell.com/heliyon/fulltext/S2405-8440(24)09543-4 

In addition to influential labs, such as EURL, that assess the application of new analytical developments within their specific fields, method developers in instrument companies must ensure that the performance statement about their devices is not countered by poor chromatography, for which they bear no responsibility.

LCTech GmbH has made a name for itself in automated sample preparation for the analysis of mycotoxins, PFAS, and other multicomponent analyte panels. Frederik Wuppermann, Product Manager Biotechnology at LCTech, describes his experience with the inert columns in mycotoxin analysis as follows.

“The separation of aflatoxin B1, G1, B2, G2, OTA, zearalenone, deoxynivalenol, T2, H-T2, fumonisin B1 and B2 after CrossTOX cleanup in high-throughput mycotoxin analysis was very good. The shorter conditioning times were also very beneficial. I can certainly also imagine the Inert Biphenyl column as an alternative for other applications.”

In addition to these independent reports, Restek has assessed the impact of the new inert LC columns on pesticides and mycotoxins. We selected these groups of analytes because they include a variety of diverse compound chemistries and are often analyzed together in multi-class screening analyses. In such screening analyses, it is very important to ensure reliable identification and quantification of the individual components at trace levels (low LOQs), and an inert LC column can help with this by providing proper peak shape and good sensitivity. For both groups, we selected an appropriate stationary phase, analyzed the target compounds on both conventional LC columns and the new inert LC columns, and compared the chromatographic results. Here are our findings.

Pesticides

For this experiment, we compared a Raptor Inert ARC-18 column to a standard Raptor ARC-18 column. Pesticides analysis can be challenging because these panels typically contain a wide variety of compound chemistries. Phosphorylated, acidic, polar compounds, and/or metal chelating species, such as organophosphate pesticides, can react to the metal surfaces inside standard, stainless-steel analytical columns, which can degrade chromatographic performance. In contrast, the deactivated hardware in Restek’s inert LC columns significantly improved chromatographic results (Figures 1-2, Table I).

Figure 1: Analysis of pesticides on a Raptor Inert ARC-18 column without any preconditioning.

LC_EV0596

Peaks

	Peaks	tR (min)	Precursor Ion	Product Ion 1	Product Ion 2	Peak Area	Peak Height
1.	Methamidophos	1.33	142.0	94.0	125.1	428941	105189
2.	Acephate	1.55	184.0	143.0	48.9	300642	104729
3.	Omethoate	1.72	214.0	125.0	182.9	892008	337690
4.	Monocrotophos	2.21	224.1	127.0	193.1	215810	78425
5.	Dicrotophos	2.35	238.1	112.1	72.0	404916	159292
6.	Dimethoate	2.52	230.0	125.0	199.0	807805	342939
7.	Trichlorfon	2.53	257.0	108.9	220.8	173942	63266
8.	Vamidothion	2.54	288.0	146.0	118.0	1333829	547308
9.	Mevinphos isomer 1	2.55	241.9	126.9	192.9	311274	129961
10.	Mevinphos isomer 2	2.76	241.9	126.9	192.9	74030	29802
11.	Carbaryl	3.18	202.1	145.0	127.0	39671	11924
12.	Isocarbophos	3.52	291.1	231.1	121.1	33294	11941
13.	Dimethomorph isomer 1	3.96	388.2	300.9	165.1	511766	172977
14.	Dimethomorph isomer 2	4.13	388.2	300.9	165.1	877031	328826
15.	Temephos	5.70	467.1	124.9	418.9	164310	64751

Conditions

Column	Raptor Inert ARC-18 (cat.# 9314A12-T)
Dimensions:	100 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Temp.:	50 °C
Standard/Sample	LC multiresidue pesticide standard #1 (cat.# 31972)
Diluent:	Water, 0.1% formic acid
Conc.:	1 ng/mL
Inj. Vol.:	5 µL
Mobile Phase
A:	Water, 2 mM ammonium formate, 0.1% formic acid
B:	Methanol, 2 mM ammonium formate, 0.1% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.4 95 5 2.00 0.4 40 60 4.00 0.4 25 75 6.00 0.4 0 100 7.50 0.4 0 100 7.51 0.4 95 5 9.00 0.4 95 5

Detector	Shimadzu LCMS-8060
Ion Mode:	ESI+
Mode:	MRM
Instrument	Shimadzu Nexera X2

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Figure 2: Compared to standard LC columns, Raptor Inert LC columns provide increased peak area and height for pesticides.

LC_EV0591

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LC_EV0593

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Table I: Pesticide peak areas and heights were up to 2X higher on Raptor Inert LC columns compared to standard columns.

Compound	Peak Area			Peak Height
	Stainless Steel	Inert	Areas Ratio (Inert/Stainless Steel)	Stainless Steel	Inert	Height Ratio (Inert/Stainless Steel)
Methamidophos	254969	428941	1.68	52553	105189	2.00
Acephate	168776	300642	1.78	58418	104729	1.79
Omethoate	579502	892008	1.54	216157	337690	1.56
Monocrotophos	140095	215810	1.54	51402	78425	1.53
Dicrotophos	340978	404916	1.19	135380	159292	1.18
Dimethoate	461156	807805	1.75	188746	342939	1.82
Trichlorfon	84233	173942	2.07	34793	63266	1.82
Vamidothion	913264	1333829	1.46	354311	547308	1.54
Mevinphos isomer 1	213632	311274	1.46	82105	129961	1.58
Mevinphos isomer 2	56093	74030	1.32	29070	29802	1.03
Carbaryl	43590	39671	0.91	14563	11924	0.82
Isocarbophos	21587	33294	1.54	9062	11941	1.32
Dimethomorph isomer 1	462425	511766	1.11	166990	172977	1.04
Dimethomorph isomer 2	896109	877031	0.98	311657	328826	1.06
Temephos	98793	164310	1.66	35383	64751	1.83

Mycotoxins

In addition to pesticides analysis, we also wanted to test whether using an inert LC column could improve results for mycotoxins analysis. Because mycotoxins are reactive compounds that can have acidic, polar, or metal-chelating groups, extensive column conditioning and equilibration is often required to obtain adequate chromatography. For these experiments, we used columns with a Biphenyl stationary phase for optimal selectivity. The results shown in Figures 3-4 and Table II clearly demonstrate that peak shape and response for mycotoxins can also be improved by using an inert column.

Figure 3: Raptor Inert Biphenyl columns provide excellent peak shape for mycotoxins without additional acid passivation or mobile phase additives.

LC_FS0552

Peaks

	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion	Peak Area	Peak Height
1.	Nivalenol	0.88	10	295.1	137.1	4182	64495
2.	Deoxynivalenol	1.25	10	297.2	231.0	17346	281906
3.	Fusarenon-X	1.92	10	355.1	137.1	7668	121790
4.	15-Acetyldeoxynivalenol	3.08	10	339.2	137.1	31369	517570
5.	3-Acetyldeoxynivalenol	3.14	10	339.2	213.1	22613	296396
6.	Tenuazonic acid	4.11	10	198.1	125.0	47828	197658
7.	Altenuene	4.60	10	293.2	257.1	113850	2059699
8.	Alternariol	5.27	10	259.0	185.1	73272	1302192
9.	Ergosine	5.28	10	548.4	208.1	486620	9366601
10.	Citrinin	5.46	10	251.2	233.1	1007880	9828889
11.	Ergosinine	5.46	10	548.4	208.1	496734	8740527
12.	Fumonisin B1	5.48	10	722.5	352.3	122878	2415567
13.	Diacetoxyscirpenol	5.62	10	384.2	247.1	68139	1208825
14.	Ergotamine	5.71	10	582.4	223.2	493003	9274155
15.	Ergocornine	5.85	10	562.4	268.2	387025	7732744
16.	Ergotaminine	5.96	10	582.4	223.2	462119	9237991
17.	HT-2	6.13	10	447.2	345.1	15221	323765
18.	Ergocryptine	6.19	10	576.4	268.2	522204	11360838
19.	Fumonisin B3	6.23	10	706.4	336.2	143302	3444421
20.	Ergocristine	6.44	10	610.4	223.2	195562	4450058
21.	Fumonisin B2	6.59	10	706.4	336.2	151719	3869822
22.	Tentoxin	6.62	10	415.2	312.2	95175	2131906
23.	α-Zearalenol	6.91	10	303.1	285.1	30224	702420
24.	Ergocorninine	6.93	10	562.4	268.2	704029	14389283
25.	Aflatoxin G2	6.97	10	331.2	189.0	262824	5274353
26.	T-2	7.09	10	489.2	387.1	56535	1394735
27.	Ergocryptinine	7.18	10	576.4	268.2	778972	16765348
28.	Ergocristinine	7.40	10	610.4	223.2	1583053	32975663
29.	Aflatoxin G1	7.45	10	329.1	199.7	304389	6102959
30.	Zearalenone	7.59	10	319.2	283.1	37162	927455
31.	Alternariol monomethylether	7.62	10	273.0	199.1	31024	640689
32.	Aflatoxin B2	7.63	10	315.1	287.0	295648	5724754
33.	Aflatoxin B1	8.02	10	313.2	241.1	223520	4425821
34.	Ochratoxin A	8.25	10	404.1	239.0	190060	4411953

Conditions

Column	Raptor Inert Biphenyl (cat.# 9309A12-T)
Dimensions:	100 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Temp.:	60 °C
Standard/Sample
	Aflatoxins standard (cat.# 34121)
	Ochratoxin A standard (cat.# 34122)
Diluent:	50:50 Water:methanol
Conc.:	10 ng/mL
Inj. Vol.:	5 µL
Mobile Phase
A:	Water, 0.05% formic acid
B:	Methanol, 0.05% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.4 75 25 5.00 0.4 50 50 9.00 0.4 0 100 9.01 0.4 75 25 11.0 0.4 75 25
Max Pressure:	440 bar

Detector	Waters Xevo TQ-S
Ion Mode:	ESI+
Mode:	MRM
Instrument	Waters ACQUITY UPLC I-Class
Notes

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Figure 4: Substantial increases in peak area and height were seen for fumonisins when using a Raptor Inert Biphenyl column.

LC_FS0554

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LC_FS0556

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Table II: For mycotoxins, peak height increased up to 10X and peak area increased up to 6X when using a Raptor Inert Biphenyl column compared to a standard column.

Compound	Peak Area			Peak Height
	Stainless Steel	Inert	Areas Ratio (Inert/Stainless Steel)	Stainless Steel	Inert	Height Ratio (Inert/Stainless Steel)
Fumonisin B1	32578	122878	3.77	399544	2415567	6.05
Fumonisin B2	23427	151719	6.48	383130	3869822	10.10
Fumonisin B3	29864	143302	4.80	472279	3444421	7.29
Ergocristine	171197	195562	1.14	3865898	4450058	1.15
Ergocristinine	1393116	1583053	1.14	29212317	32975663	1.13
Ergotamine	433635	493003	1.14	8149518	9274156	1.14
Ergotaminine	397370	462119	1.16	7885403	9237991	1.17
Ergocryptine	446481	522204	1.17	9671753	11360839	1.17
Ergocryptinine	658788	778972	1.18	13680420	16765348	1.23
Ergocornine	370509	387025	1.04	7248981	7732744	1.07
Ergocorninine	590167	704029	1.19	12052359	14389283	1.19
Ergosine	445243	486620	1.09	8630932	9366602	1.09
Ergosinine	439026	496734	1.13	7820785	8740527	1.12
T-2	43286	56535	1.31	1046233	1394735	1.33
HT-2	10183	15221	1.49	216703	323765	1.49
Tentoxin	70973	95175	1.34	1577164	2131907	1.35
Ochratoxin	173686	190060	1.09	4039682	4411953	1.09
Diacetoxyscirpenol	47850	68139	1.42	846403	1208826	1.43
Fusarenone X	3865	7668	1.98	60409	121790	2.02
15-acetyl-DON	17055	31369	1.84	269862	517570	1.92
3-acetyldeoxyvinalenol	13353	22613	1.69	179204	296396	1.65
Aflatoxin G2	171597	262824	1.53	3429501	5274354	1.54
Aflatoxin G1	224058	304389	1.36	4607959	6102959	1.32
ZON	25617	37162	1.45	656915	927455	1.41
Aflatoxin B2	159389	295648	1.85	3462489	5724754	1.65
Aflatoxin B1	265935	223520	0.84	5335576	4425821	0.83
Alpha-zearalenol	16202	30224	1.87	382092	702420	1.84
Deoxynivalenol	6935	17346	2.50	117927	281906	2.39
Nivalenol	1790	4182	2.34	25276	64495	2.55
Altenuene	63224	113850	1.80	1187958	2059700	1.73
Alternariol monomethyl ether	19537	31024	1.59	428922	640689	1.49
Alternariol	48204	73272	1.52	837410	1302192	1.56
Citrinin	499900	1007880	2.02	5031182	9828890	1.95
Tenuazonic acid	21503	47828	2.22	89293	197658	2.21

While our pesticides and mycotoxins experiments showed that columns made with inert hardware can improve chromatographic performance, the independent results from Carrera and Wuppermann are even more compelling. Based on the experiences of these well-regarded scientists, in addition to our own testing, it seems clear that Restek’s Inert LC columns have many potential benefits to laboratories analyzing pesticides and mycotoxins. It will be interesting to see what other analyses may be improved as well!

Products Mentioned

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Catalog No. 9309A12-T

Raptor Inert Biphenyl, 2.7 µm, 100 x 2.1 mm HPLCカラム

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Catalog No. 34122

Ochratoxin A Standard, 10 μg/mL, Acetonitrile, 1 mL/ampul

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Catalog No. 34121

Aflatoxins (B1, B2, G1, G2) Standard, 10 µg/mL, Acetonitrile, 1 mL/ampul

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Catalog No. 31972

LC Multiresidue Pesticide Standard #1 Organophosphorus Compounds, 100 µg/mL, Acetonitrile, 1 mL/ampul

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Catalog No. 9314A12-T

Raptor Inert ARC-18, 2.7 µm, 100 x 2.1 mm HPLCカラム

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Author

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  Hansjörg Majer, PhD

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