Agilent Syed Salman Lateef Agilent Technologies, Inc. Bangalore, India 8 6 4 2 1.191 1.678 12.46 2.5 5 7.5 1 12.5 ( C) ( ) Agilent Poroshell EC-C18 Agilent 126 Infinity LC (LOD).2 µg/ml 3 9 % Agilent 129 Infinity LC (UHPLC) UHPLC LOD 5 1
(DHA) 1 4 6 µg/ml 2,3 4 DHA AOAC 967.22 DHA C UVDHA 22 nm ph 244 265 nm 5 AOAC 994.11 UV UV HPLC Milli Q (Millipore Elix 1 ) Lab-Scan ( ) Fluka () o- Fluka () Sigma-Aldrich () Agilent 126 Infinity LC Agilent 126 Infinity (G1312B) Agilent 126 Infinity (G1367E G133B) Agilent 126 Infinity (G1316A) Agilent 126 Infinity (G4212B) 1-mm Max- Light UHPLC Agilent 129 Infinity LC Agilent 129 Infinity (G422A) Agilent 129 Infinity (G4226A G133B) Agilent 129 Infinity (G1316C) Agilent 129 Infinity (G4212A) 1-mm Max- Light : Agilent Poroshell 12 EC-C18 4.6 x 1 mm 2.7µm ( 697975-32) : Agilent ChemStation B.4.2 2
UHPLC 1 A 5, µg/ml (ppm) 5, ppm 1 ppm 1 A 2 A ph 2.5 ph o- 5 ml ph 2.5 1879 g 5 Agilent.2 µm ( 5185-583) A 5 µl 6 (RT) (RSD) 3 (LOD) (LOQ) Agilent 126 Infinity LC Agilent 129 Infinity LC TCC 2 ºC 2 ºC 4 Hz 4 Hz DAD (nm) 21. 23. 243.5 21. 23. 243.5 1 mm 1 µl 1 mm 1 µl 4 ºC 4 ºC A 1 Agilent 126 Infinity LC Agilent 129 Infinity LC (µg/ml) (µg/ml) (µg/ml) 1 1 55.2 2 45 6 1 3 63 65 2 4 9 7 3 5 18 75 5 6 144 8 1 7 162 85 2 8 18 9 35 9 225 5 2 3 2 mm (KH 2 PO 4 ) o- ph 2.5 B 6 % - 4 % 2 mm (KH 2 PO 4 ) o- ph 2.5 6 % - 4 % ( ) %B ( ) %B 5 5 2 5.5 5 2.1 25.6 25 13. 25 3. 25 13.1 9 3.1 7 18. 9 3.9 7 18.1 5 4. 5 25. 5 5. 5 1. ml/min 1.5 ml/min 5 µl 3. 4 µl 5. 3
ph 2.5 C 4 ( 2 % TCC 5 % 5 % 3 %) C 18 ppm 7, ppm 5 ppm 7 3 3 HPLC UHPLC LOD LOQ RT RSD C A - Poroshell EC-C18 Agilent Poroshell EC-C18 TCC B 6 % 4 % ph 2.5 243.5 nm 21. nm 23. nm 4 ºC Margolis 6 22 89 % Margolis - 4 % 3 8 6 4 2 1.191 1.678 MS ( 519-228) ALS ( 5182-716) - ALS ALS 4 ºC 3 16 3 % 1 % MS 1 Agilent 126 Infinity LC B 25 % 13 12.46 2.5 5 7.5 1 12.5 1 Agilent Poroshell 12 EC-C18 23 nm 4
(LOD) (LOQ) / (S/N) 3 LOD S/ N 1 LOQ S/N LOD LOQ LOD S/N = 3.5 µg/ ml LOQ S/N = 16.2 µg/ml 3 LOQ 6 3 3 LOD LOQ 1.2 1.8.6.4.2 LOQ.2 µg/ml 12.2 12.3 12.4 12.5 12.6 12.7 12.8 2.2 µg/ml ( 1 ng) (LOQ ) S/N 16 mau 35 3 25 2 15 y = 14.11x + 15.542 R 2 =.9998 12.9 13 1 5 5 1 15 2 25 (ppm) 3 1 µg/ml 225 µg/ml Sl : 1 LOD µg/ml S/N LOQ µg/ml S/N (µg/ml) R 2 n= 6 1 225.9998 9 L1=87 % (98 % 1 %) 2 55 9.9995 8 99 % 11 % 3.5 3.2 16.2 5 1 9 L1 = 16 % 96 11 % 3 5
(RT) RSD (%) RSD 1 (L1) 1.9 % RT RSD.13 % RT RSD RSD 4 RSD (%) 2.5 2 1.5 1 18 µg/ml 7, µg/ml 5 µg/ml 4 (TCC ) 7 6 5 % 3 %.5 1 2 3 4 5 6 7 8 9 1 4 6 RSD (%) 6
: 1. ml/min 2 % : 1.2 ml/min :.98 ml/min 23 nm % % RT % % % RT % % RT 6.2 2.1. 2.2 2.2 3.4 1.8 3.3 1.8.6 1.2 1.3.5 1.7 TCC : 2 ºC 5 % : 21 ºC : 19 ºC : 5 µl 5 % : 5.25 µl : 4.75 µl 7.6.4.1.1.6 2. 1.4 18.1.5.7.4.9 2.7 1.5 2.4.2 1.6 5.6.3 4.3.9 8.2..5 5..2 6.1.2 : 21. 23. 243.5 nm 3 nm : 213. 233.3 246.5 nm : 27. 227. 24.5 nm 2.5.1.1 5.6.2 5.2.5 3.3.1.8 3.3.1 3.4.3 4 4 2 % RT 5 % RT TCC UV DAD 3 (3 µg) (4, µg) (2 µg) (o- ph 2.5 ) (6 µg) (8, µg) (4 µg) (%) 1 3 91 12 98 6 5 3 (5 3 ) / 3 5 243.5 nm 3 9 % 7
3 (O- 1 O- 2 O- 3 ) ( 6) 3 O- 1 111 µg/ml 145 µg/ml O- 1 O- 3 E3 E33 ( ) 2 5 2.25 µg/ml 6 2 6 2 6 2 6 2 1.191 1.189 1.189 1.682 1.68 1.68 1.19 1.698 2 4 µg/ml 6 8 µg/ml 1 12.448 O- 1 12.512 O- 2 O- 3 5 23 nm 3 12 12.461 2 _ 2 2 _ 2 2 _ 2 2 _ 2 µg/ml O- 1 145 ± 2 8895 ± 21.62 ±.7 O- 2 93 ± 3 8188 ± 43 ± O- 3 35.4 ±.3 316 ± 8 2.25 ±.6 6 3 ( ) 23 nm 12.448 12.512 12.461 11 12 13 14 8
UHPLC HPLC Agilent 129 Infinity LC UHPLC HPLC 25 UHPLC 6 5 5 68 % UHPLC B 9 % 7 % 9 % 7 % Agilent 129 LC Infinity LOD.5 µg/ml LOQ.2 µg/ml (S/N =16) Agilent 126 Infinity LC 7 mau 4 3 2 1 16 14 12 1 8.389.285 2.75 1 2 3 6 Agilent Poroshell 12 EC-C18 3. x 75 mm 2.7-µm UHPLC 6 y = 5.9417x - 4.2859 R 2 =.9997 4 2 5 1 15 2 25 (ppm) 7 Agilent 129 Infinity LC 1 µg/ml 225 µg/ml ( 5 ng 1125 ng) 9
HPLC RT RSD (%) 8 RSD (%) 2.5 % RT RSD.5 % 2.5 2 Agilent Poroshell 12 EC-C18 25 9 % Agilent 129 Infinity LC 5 UHPLC RSD (%) 1.5 1.5 1 2 3 4 5 6 7 8 9 1 8 3 RSD (%) UHPLC 6 1
1. INCHEM chemical safety information for intergovernmental organization: http://www.inchem.org/ documents/cicads/cicads/cicad26. htm#subsectionnumber:5.1.1 2. US FDA, Food contamination and adultration: Data on Benzene in Soft Drinks and Other Beverages Data through May 16, 27 http:// www.fda.gov/food/foodsafety/ FoodContaminantsAdulteration/ ChemicalContaminants/Benzene/ ucm55815.htm 3. L.K Gardner, G.D Lawrence, Benzene Production from Decarboxylation of Benzoic Acid in the Presence of Ascorbic Acid and a Transition-Metal Catalyst, J. Agric. Food. Chem, 41: 693-695, 1993. 4. K. Zerdine; M.L. Rooney; J Vermue, The Vitamin C Content of Orange Juice packed in an Oxygen Scavenger Material, Food Chemistry, 82: 387-395, 23. 5. L. Novakova; P. Solich; D. Solichova, HPLC methods for simultaneous determination of ascorbic acid and dehy-droascorbic acids, Trends in Analytical Chemistry, 27: 942-958, 28. 6. S.A. Margolis; E. Park, Stability of Ascorbic Acid in Solutions Stored in Autosampler Vials, Clinical Chemistry, 47: 1463-1464 21. 11
www.agilent.com/chem/lc:jp Agilent Technologies, Inc., 211 Published in Japan, September 1, 211 599-872JAJP