Documentation

Method Abstract 223

MA 223 / ANALYSIS OF SUGARS IN FEEDS

BY HPLC WITH POST-COLUMN DERIVATIZATION AND FLUORESCENCE DETECTION

The types and amounts of sugar in animal feeds are as important as the amount of protein, minerals and fats in the determination of nutritive value. We developed a simple and sensitive HPLC method for analyzing six sugars in animal feeds – Sucrose, Fructose, Glucose, Galactose, Maltose and Lactose. Post-column derivatization reagents convert reducing and non-reducing sugars into fluorescent derivatives, which greatly improves the sensitivity and selectivity of the detection.

The blends of feed examined varied from grains/vegetable products (live stock feeds) to meat/vegetable products (pet food).
 

METHOD

Standards Preparation:

Mix 2.5 g of feed sample with 50 mL of water. Heat using a water bath while constantly mixing for 1 hour at 65 °C. Centrifuge and filter through 0.45 um filter.

Analytical Conditions

Column: Carbohydrate column, 4.6×150 mm
Temperature: 30 ºC
Flow Rate: 1 mL/min
Mobile Phase: Acetonitrile/Water
Injection Volume: 10 uL – 50 uL

Post-Column Conditions

Post-Column System: Pinnacle PCX or Vector PCX
Reactor Volume: 1.4 mL
Temperature: 130 °C
Reagent 1: Guanidine hydrochloride 60 mM in 200 mM Boric acid adjusted to pH 11.5 with KOH
Reagent 2: 1.5 mM periodic acid adjusted to pH 11.5 with KOH
Flow Rate: 0.15 mL/min each reagent
Detection: FLD; λex: 325 nm, λem: 465 nm
 

HPLC GRADIENT

TIME (Min)

WATER, %

ACN, %

0.0

20

80

20.0

 20  80

20.1

 50  50

30.0

 50  50

30.1

 20  80


Calibration

A quadratic calibration curve with correlation > 0.999 is observed for monosaccharides such as Fructose, Glucose and Galactose. A linear calibration curve with correlation > 0.999 is observed for disaccharides such as Maltose, Lactose and Sucrose. Examples of calibration curves presented in Fig. 1 and Fig. 2.

M_223a

HPLC GRADIENT

 

FRUCTOSE

GLUCOSE GALACTOSE SUCROSE MALTOSE LACTOSE
FEED MATRIX 1
Content in feed, % 0.54 0.52 0.09 4.02 1.12 ND*
Spike Concentration, % 0.60 0.58 0.60 2.02 0.58 0.59
Recoveries, n=3, % 105 107 110 91 103 114
Spike Concentration, % 1.21 1.20 1.19 4.05 1.22 1.21
Recoveries, n=3, % 108 106 110 85 85 103
FEED MATRIX 2
Content in feed, % 0.23 0.46 ND* 3.21 0.59 ND*
Spike Concentration, % 0.42 0.57 0.56 2.50 0.57 0.58
Recoveries, n=3, % 98 101 104 106 106 107
Spike Concentration, % 0.81 1.14 1.12 4.80 1.11 1.15
Recoveries, n=3, % 103 101 101 102 102 106
FEED MATRIX 3
Content in feed, % 0.14 0.11 ND* 0.51 0.02 ND*
Spike Concentration, % 0.38 0.56 0.57 2.40 0.55 0.56
Recoveries, n=3, % 95 101 116 102 95 101
FEED MATRIX 4
Content in feed, % 0.17 0.13 ND* 1.35 0.21 ND*
Spike Concentration, % 0.41 0.57 0.58 2.43 0.56 0.56
Recoveries, n=3, % 92 97 116 101 95 102

*Not Detected
 

M_223b
   
  M_223c