Agilent Determination of aflatoxins with Agilent 1290 Infinity LC Agilent 1260 Infinity Binary LC Agilent ZORBAX RRHT 1.8 m columns by FLD after electrochemical derivatization with a Coring C
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1. 3900 Alla G2 Figure 1 10 777 Alla 31 15 412 Afla B1 M m z a 5 Separation on a standard LC column Eurospher Knauer Germany 250 mm x 4 6 mm 5 pm 1 mL min 30 C Experimental Analysis of aflatoxins can be achieved by using a fast LC configuration Table 1 Experimental conditions Eluent Wasser Acetonitrile Methanol 65 Nitric Acid KBr 600 200 200 100 uL 119 mg L pH 2 79 Detection FLD Ex 362 nm Em 435 nm Flow 1 0 mL min Injection volume 20 uL Temp 40 C The setup for post column derivatiza tion was preset according to the recommendations of Coring for analysis of aflatoxines with conventional HPLC Results and Discussion The first run of a standard was per formed in a conventional 250 mm col umn Figure 1 The parameters were transferred to a 100 mm x 4 6 mm RRHT column filled with 1 8 pm particles using the Method Translator Figure 2 shows the results of the afla toxines standard analysis The results illustrate comparable resolution but the shorter column reduced analysis time Table 2 Additional evidence of a successful transfer was the determination of the aflatoxines in food samples such as marzipan or hazelnuts to verity the influence of the matrix All aflatoxins are well separated resolu tion gt 1 5 for all peaks with reduced analysis time of 8 0 minutes In Figure 3 the separation of the aflatoxins in a hazelnut sample2. G1 and G2 show absorption by UV but the detection limits of food control are so low that UV detection is not sensitive enough To improve detection sensitivity the aflatoxins can be post column deriva tized with a Coring Cell After post column electrochemical bromation of mycotoxins sensitivity can be dramati cally enhanced using fluorescence detection The influence of matrix com ponents is also reduced reaching lower limits of detection Post column derivatization requires a four second reaction time between Coring Cell and detector This is usually achieved by low flows and wide capil laries This Application Note addressed the following question can post column derivatization be combined with rapid resolution LC to achieve better results In the analysis of aflatoxines even in complex matrices such as marzipan and hazelnuts Agilent 1290 Infinity LC G4220A Agilent 1290 Infinity Binary Pump with Integrated Vacuum Degasser G4226A Agilent 1290 Infinity Autosampler G1316C Agilent 1290 Infinity Thermostatted Column Compartment G1321A Agilent Fluorescence Detector Table 1 Agilent 1260 Infinity Binary LC G1312B Agilent 1260 Infinity Binary Pump with Vacuum Degasser G1367C Agilent 1260 Infinity Autosampler G1316B Agilent 1260 Infinity Thermostatted Column Compartment G1321A Agilent 1260 Infinity Fluorescence Detector Configuration of the Agilent 1290 Infinity LC system and Agilent 1260 Binary LC system
3. Determination of aflatoxins with Agilent 1290 Infinity LC Agilent 1260 Infinity Binary LC and Agilent ZORBAX RRHT 1 8 um columns by FLD after electrochemical derivatization with a Coring Cell Application Note Author Detlef Wilhelm Analox GmbH amp Co KG Fuerstenwalde Germany Abstract The determination of contaminants in foods requires high throughputs and high sensi tivities With the Agilent 1290 Infinity LC system and the Agilent 1260 Binary LC sys tems it is possible to transfer methods with low outputs to faster applications The determination of aflatoxins B1 B2 G1 and G2 by HPLC and post column derivatization for fluorescence detection can be accelerated by using shorter Agilent ZORBAX RRHT 1 8 um columns instead of conventional 5 um columns High resolution was achieved despite post column derivatization with the Coring Cell and related band broadening Sample throughput was increased by a factor of gt 2 with analysis times of about 10 min including column flushing Ee Agilent Technologies Introduction Aflatoxins are naturally occurring myco toxins produced by certain molds grow ing on some food crops during produc tion and storage Aflatoxin B1 Figure 5 is the most dangerous because It Is carcinogenic and very small amounts can be toxic for humans For that rea son foods must be monitored because contamination by various mildews pro ducing aflatoxins can easily occur Aflatoxins B1 B2
4. a complex matrix com pared to marzipan in Figure 4 shows a low influence of matrix components By analyzing strong matrix contaminated samples it is proven that a short flush ing gradient with acetonitrile to 80 in B can be used to flush the column Conclusions All Agilent systems show excellent per formance regarding aflatoxin analysis Methods with separations of conven tional materials can be easily trans ferred to achieve shorter analysis times with comparable chromatographic reso lution even with the robust 4 6 mm columns filled with 1 8 um particles Both Agilent 1260 Infinity Binary LC and the Agilent 1290 Infinity LC showed significant time reduction resulting Figure 2 3 743 Aflatoxine 62 r a x o a a 755 127 Aflatoxine B2 Separation with Agilent SB C18 100 mm x 4 6 mm 1 8 um 40 C 1 mL min Figure 3 Determination of aflatoxins in hazelnuts Figure 4 Determination of aflatoxins in marzipan 3 750 Aflatoxine G2 9 Aflatoxine G1 6 259 Aflatoxine nn 5 151 Aflatoxine B2 6 259 Aflatoxin E1 4 441 Aflatoxine G1 5 151 Aflatoxine B2 from the transfer to shorter high reso Mycotoxin Retention time min lution columns Aflatoxin G2 3 743 Aflatoxin G1 4 482 The extended power range of the Agilent 1290 Infinity LC systems allows s l aa the use of RRHD columns Therefore an ZOD 6 20 Agilent StableBond 100 mm x 2 1 mm Table 2 1 8 um particle co
5. lumn can be used at the same flow rate producing a shorter cycle time with the same resolution References 1 Figure 5 Structure of aflatoxin B1 2 List of aflatoxin compounds and retention times User Manual of the Coring Cell Coring System Diagnostix GmbH Gernsheim Germany Aflatoxins in nuts survey http www food gov uk news newsar chive 2004 jun aflatoxinsurvey www agilent com chem infinity Agilent Technologies Inc 2010 July 1 2010 Publication Number 5990 6167EN ie Agilent Technologies
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