**5. Optimization the conditions of chromatographic separation**

The method of high performance liquid chromatography (HPLC) with flouorometric detection or a diode array is characterized by sufficient selectivity and sensitivity required to determine feed additives. Each of the developed and verified procedures includes precisely defined stages of hydrolysis and extraction which make it possible to determine the total or added analyte in a sample. Chromatographic separation of the analyzed mixture is affected by the properties of chromatographic arrangement. The developed analytical methods took advantage of chromatography in a regular and reversed arrangement of phases. In the reversed phase methanol and water or acetonitrile and water were used. In the standard arrangement the mobile phase was hexane or chloroform. In the majority of the developed methods presented in this chapter isocratic elution was applied, except for methionine hydroxy analog where gradient elution was used. Adequate separation was achieved through the use of ODS column with 18 atoms of carbon in the chain (C18) and columns with 8 atoms of carbon (C8) in the alkyl chain in chromatography with reversed phase (RP). In chromatography with the normal phase arrangement columns filled with silica gel were used. The identification and content of analyte was examined with the method of absolute calibration (with external model), analyzing separately the sample and the model and identifying the peaks with the help of retention values, comparing retention time of the identified substance with the retention time of a standard, chromatographed in identical conditions. Examples of chromatograms for the standard extract and the sample of the examined analyte (riboflavin) with the use of fluorescent detector are presented below (Fig.1, Fig.2).

174 Chromatography – The Most Versatile Method of Chemical Analysis

A sample for a quantitative analysis should be prepared in such a way that the isolation of a selected analyte and removal of interfering substances is possible. The condition necessary for adequate quantitative determination in liquid chromatography is eliminating any possibility of coelution and minimization the drift of a basic line. Extractions of the analyzed substances were done classically by shaking the sample with a solvent. In case of vitamins B1 and B2 and carotenoids, such as canthaxanthin and apocarotenic acid ester, extraction was performed in an ultrasonic bath. In order to purify the extract, aluminum oxide (e.g. canthaxanthin), celite and anhydrous sodium sulphate (e.g. vitamin K3) were used and PTFE and Nylon (PA) (0.45 μm or 0.20 μm) syringe filters were applied before injecting on the chromatographic column. Syringe (hydrophobic) PTFE filters are used in case of solutions with high acid and base content, whereas nylon (hydrophilic) filters are used with aqueous and organic solutions. Filtration of extracts is necessary as it prolongs durability of a column due to eliminating permanent contamination which blocks the column's intake and increases back pressure. If needed, the analyte may be concentrated by evaporating the solvent. When there is a risk that the studied analyte becomes oxidized, evaporation is done

The removal of gelatin-and-sugar beadlets protecting vitamins A and D3 is done at the stage of saponification and transforming the vitamins into alcoholic forms. Douša & Břicháč [16] demonstrated that saponification in standard conditions did not affect the results of analyses. In case of canthaxanthin enzymatic hydrolysis through adding trypsin and pepsin is used. While determining the total content of thiamine and/or riboflavin in feedingstuffs (endogenic and added) at the stage of preliminary preparation an ultrasonic bath and also

The method of high performance liquid chromatography (HPLC) with flouorometric detection or a diode array is characterized by sufficient selectivity and sensitivity required to determine feed additives. Each of the developed and verified procedures includes precisely defined stages of hydrolysis and extraction which make it possible to determine the total or added analyte in a sample. Chromatographic separation of the analyzed mixture is affected by the properties of chromatographic arrangement. The developed analytical methods took advantage of chromatography in a regular and reversed arrangement of phases. In the reversed phase methanol and water or acetonitrile and water were used. In the standard arrangement the mobile phase was hexane or chloroform. In the majority of the developed methods presented in this chapter isocratic elution was applied, except for methionine hydroxy analog where gradient elution was used. Adequate separation was achieved through the use of ODS column with 18 atoms of carbon in the chain (C18) and columns with 8 atoms of carbon (C8) in the alkyl chain in chromatography with reversed phase (RP). In chromatography with the normal phase arrangement columns filled with silica gel were

**5. Optimization the conditions of chromatographic separation** 

**4. Extraction and extract purification** 

in neutral gas, e.g. nitrogen or argon.

enzymatic hydrolysis (taka-diastase) are applied.

**Figure 1.** Characteristic chromatograms of riboflavin: chromatogram of standard solution

**Figure 2.** Characteristic chromatograms of riboflavin: chromatogram of premix extract

### **6. Selected methods of testing feed additives**

Selected methods of testing feed additives presented below were validated with the help of a high pressure liquid chromatograph (Dionex P-680) with fluorescence detector (Dionex RF 2000) or with a diode array.

## **Vitamin B1 (Thiamine)**

The following procedure used for determination of thiamine in premixes and compound feeds was elaborated on the basis of the article published by Rubaj *et al*. [18].

Using High Performance Liquid Chromatography (HPLC) for Analyzing Feed Additives 177

content was determined by high performance liquid chromatography (HPLC) with

**Reagents and Solvents.** All reagents and solvents should be of analytical grade: methanol for HPLC; sulphuric acid, 0.1 mol/l; sulphuric acid, 30%; sodium hydroxide, 0.5 mol/l; sodium acetate, 2mol/l; acetic acid, 99.5%; citric acid; taka-diastase, 10% suspension; vitamin

Riboflavin was extracted from the examined feed sample with 0.1M sulphuric acid, and that solution was boiled for 15 min. at temperature from 110°C to 120°C. After cooling to the room temperature, the whole volume of hydrolysed sample was transferred to a 100 ml measuring flask. Next taka-diastase suspension was added to the flask, which was then placed into a water bath at 45°C for 20 min. The enzymatic reaction was stopped by adding sulphuric acid. The sample solution was next chilled to room temperature, and the volume was corrected to 100 ml by adding 0.1 mol/l sulphuric acid. Afterwards, samples were mixed and filtrated. Extract clean-up was done by adding methanol to the sample and

Mobile phase Methanol and citric acid 0.2 mg/l (30:70 v/v). That solution was

Vitamin B2 is sensitive to light, hence all the activities were conducted without any access of

The following procedure for determination of canthaxanthin in premixes and compound

The principle of this method is based on the hydrolysis of a powdered formulation of canthaxanthine with trypsin and pepsin in water solution of ammonia and its purification on the aluminium oxide filled column. The canthaxantine content is determined by high performance liquid chromatography (HPLC) in normal phase with usage of DAD detector.

**Apparatus**: autoclave, ultrasound bath, HPLC set with fluorescence detector.

filtration through syringe filter before injection on the column.

mixed with methanol with ratio 1:1

Detector Fluorescence, Ex λ= 453, Em λ=521

Calculation External standard, peak area, linear regression

day light (by using amber glass flask or flask covered by aluminum foil).

feeds was elaborated on the basis of the article published by Rubaj et al. [22].

Column 25 cm x 4.6 mm

Stationary phase C18

Column temperature 25 ºC Flow rate 0.8 ml/min Injection 20 μl

reversed-phase and usage of fluorescence detection.

B2 standard.

**Procedure** 

**Chromatography** 

**Special Comment** 

**Canthaxanthin** 

**Principle** 

## **Principle**

Vitamin B1 is extracted with hydrochloric acid of 0.1 mol/l and next oxidized to thiochrome and marked with the use of high performance liquid chromatography (HPLC) with a fluorescence detection.

**Reagents and Solvents.** All reagents and solvents should be of analytical grade: chloroform; methanol; hydrochloric acid, c=0.1 mol/l; trichloroacetic acid, 50%; sodium hydroxide, 15%; water saturated isobutanol; potassium hexacyanoferrate (III) ; vitamin B1 standard, takadiastase, sodium acetate.

**Apparatus:** laboratory shaker, centrifuge, water bath with Allin condenser, HPLC set with fluorescence detector.

## **Procedure**

Thiamine was extracted from the examined feed sample with 0.1 M hydrochloric acid at 100°C for 30 minutes. In case of compound feedingstuffs 10% taka-diastase solution was added to the samples, and then samples were incubated at 37°C for 17 hours. Afterwards thiamine was oxidized to thiochrom by 1% alkaline K3Fe(CN)6.

## **Chromatography**


## **Special Comment**

This method was applied for the quantification of total content of thiamine in compound feedingstuffs as well as added thiamine in the form of hydrochloric or nitrate salt.

### **Vitamin B2 (Riboflavin)**

The following procedure for determination of riboflavin in premixes and compound feeds was elaborated on the basis of the article published by Rubaj et al. [17].

### **Principle**

Riboflavin was extracted from a feed sample in autoclave with 0.1M sulphuric acid. The ester bonds with phosphoric acid were hydrolyzed by the Taka-diastase enzyme. Riboflavin content was determined by high performance liquid chromatography (HPLC) with reversed-phase and usage of fluorescence detection.

**Reagents and Solvents.** All reagents and solvents should be of analytical grade: methanol for HPLC; sulphuric acid, 0.1 mol/l; sulphuric acid, 30%; sodium hydroxide, 0.5 mol/l; sodium acetate, 2mol/l; acetic acid, 99.5%; citric acid; taka-diastase, 10% suspension; vitamin B2 standard.

**Apparatus**: autoclave, ultrasound bath, HPLC set with fluorescence detector.

## **Procedure**

176 Chromatography – The Most Versatile Method of Chemical Analysis

The following procedure used for determination of thiamine in premixes and compound

Vitamin B1 is extracted with hydrochloric acid of 0.1 mol/l and next oxidized to thiochrome and marked with the use of high performance liquid chromatography (HPLC) with a

**Reagents and Solvents.** All reagents and solvents should be of analytical grade: chloroform; methanol; hydrochloric acid, c=0.1 mol/l; trichloroacetic acid, 50%; sodium hydroxide, 15%; water saturated isobutanol; potassium hexacyanoferrate (III) ; vitamin B1 standard, taka-

**Apparatus:** laboratory shaker, centrifuge, water bath with Allin condenser, HPLC set with

Thiamine was extracted from the examined feed sample with 0.1 M hydrochloric acid at 100°C for 30 minutes. In case of compound feedingstuffs 10% taka-diastase solution was added to the samples, and then samples were incubated at 37°C for 17 hours. Afterwards

This method was applied for the quantification of total content of thiamine in compound

The following procedure for determination of riboflavin in premixes and compound feeds

Riboflavin was extracted from a feed sample in autoclave with 0.1M sulphuric acid. The ester bonds with phosphoric acid were hydrolyzed by the Taka-diastase enzyme. Riboflavin

feedingstuffs as well as added thiamine in the form of hydrochloric or nitrate salt.

thiamine was oxidized to thiochrom by 1% alkaline K3Fe(CN)6.

Detector Fluorescence, Ex λ= 365, Em λ=435

Stationary phase LichroCart 250-4, Lichrospher100 NH2 (5μm) Mobile phase Chloroform and methanol, 90+10 (v/v)

Calculation External standard, peak area, linear regression

was elaborated on the basis of the article published by Rubaj et al. [17].

Column 25 cm x 4.6 mm

Column temperature 25 ºC Flow rate 2.0 ml/min Injection 20 μl

feeds was elaborated on the basis of the article published by Rubaj *et al*. [18].

**Vitamin B1 (Thiamine)** 

fluorescence detection.

diastase, sodium acetate.

fluorescence detector.

**Chromatography** 

**Special Comment** 

**Principle** 

**Vitamin B2 (Riboflavin)** 

**Procedure** 

**Principle** 

Riboflavin was extracted from the examined feed sample with 0.1M sulphuric acid, and that solution was boiled for 15 min. at temperature from 110°C to 120°C. After cooling to the room temperature, the whole volume of hydrolysed sample was transferred to a 100 ml measuring flask. Next taka-diastase suspension was added to the flask, which was then placed into a water bath at 45°C for 20 min. The enzymatic reaction was stopped by adding sulphuric acid. The sample solution was next chilled to room temperature, and the volume was corrected to 100 ml by adding 0.1 mol/l sulphuric acid. Afterwards, samples were mixed and filtrated. Extract clean-up was done by adding methanol to the sample and filtration through syringe filter before injection on the column.

### **Chromatography**


### **Special Comment**

Vitamin B2 is sensitive to light, hence all the activities were conducted without any access of day light (by using amber glass flask or flask covered by aluminum foil).

### **Canthaxanthin**

The following procedure for determination of canthaxanthin in premixes and compound feeds was elaborated on the basis of the article published by Rubaj et al. [22].

### **Principle**

The principle of this method is based on the hydrolysis of a powdered formulation of canthaxanthine with trypsin and pepsin in water solution of ammonia and its purification on the aluminium oxide filled column. The canthaxantine content is determined by high performance liquid chromatography (HPLC) in normal phase with usage of DAD detector.

**Reagents and Solvents:** trypsin 200 FIP – U/g; pepsin 700 FIP – U/g; ammonia; n – hexane; diethyl ether; 99.8% ethyl alcohol; acetone; aluminum oxide, neutral, activity 1; canthaxanthin standard; chloroform. All reagents and solvents should be of analytical grade.

Using High Performance Liquid Chromatography (HPLC) for Analyzing Feed Additives 179

**Chromatography** 

**Special Comment** 

MHA retention.

recovery and uncertainty.

external calibration was used.

Column 25 cm x 4.6 mm Stationary phase C18, LiChrospher

Detector UV, λ=214 nm

Column temperature 25 °C Flow rate 0.8 ml/min Injection 20 μl

Mobile phase Eluent 1 : acetonitrile - phosphoric acid 10+90 (v/v) Eluent 2 : acetonitrile - phosphoric acid 23+77 (v/v)

Calculation External standard, peak area, linear regression


**7. Validation parameters of the methods used to analyze feed additives** 

A significant element in verifying a chemical method, including chromatography, is its validation. Validation is a confirmation through examining and presenting some objective evidence that some particular requirements regarding the intended application have been fulfilled. The basic validation parameters include: calibration linearity, the limit of detection and quantitative determination, precision (repeatability, indirect precision, reproducibility),

Calibration linearity is defined as a relationship between the readings of the measuring device and the concentration of a particular component, in conformity with the regression equation: bx + a = y. The measure of linearity is Pearson's linear correlation coefficient (r) for parameters with regular distribution. The scale presented below is adopted to estimate the correlation coefficient: 0.0-0.2: very weak relationship; 0.2-0.4: weak relationship; 0.4-0.6:

In case of feed additives discussed in the present chapter, determined with HPLC methods,

In case of chromatographic methods the value of limit of detection (LOD) may be determined on the basis of the obtained chromatogram of blanc sample, as the threefold value of a noise signal. To do this, it is necessary to determine the level of noise, by measuring on the chromatogram the range of signal change near retention time of examined

moderate relationship; 0.6-0.8: strong relationship; 0.8-1.0: very strong relationship.

**Apparatus**: ultrasonic bath; vacuum rotary evaporator, HPLC set with Array's diode detector.

## **Procedure**

The sample is hydrolyzed with an aqueous solution of ammonia at the presence of trypsin and pepsin following extraction with ethyl alcohol and diethyl ether. Purification occurs on the aluminum oxide filled column. The extract prepared in this way should be evaporated, dissolved in the mobile phase, filtered through syringe filters and dosed on the column.

## **Chromatography**


## **Methionine hydroxy analog (MHA)**

The procedure of analyzing methionine hydroxy analog was developed on the basis of the work by Matyka *et al.* [25] and the official VDLUFA method [8].

## **Principle**

Methionine hydroxy analog is extracted from the sample by 10% acetonitrile, and next hydrolyzed with potassium hydroxide and determined by high performance liquid chromatography (HPLC) with reversed phase and UV detection.

**Reagents and Solvents:** acetonitrile; orthophosphoric acid; solution for extraction: acetonitrile - water 10+90 (V/V); solution for hydrolysis: 50% potassium hydroxide (w/v); phosphoric acid, 0.01mol/l.

**Apparatus**: centrifuge, HPLC set, diode array detector.

### **Procedure**

Extract methionine hydroxy analog from the feed, with the use of 10% acetonitrile. After centrifuging, perform hydrolysis with potassium hydroxide and next with a solution of orthophosphoric acid. Filter the supernatant through syringe filters and inject on the column.


## **Chromatography**

178 Chromatography – The Most Versatile Method of Chemical Analysis

detector.

**Procedure** 

column.

**Principle** 

**Procedure** 

column.

**Chromatography** 

Column 4.6 x 250 mm Stationary phase LiChrospher Si 60

Detector DAD λ=446 nm

**Methionine hydroxy analog (MHA)** 

phosphoric acid, 0.01mol/l.

Column temperature 25 ºC Flow rate 1.3 ml/min Injection 20 μl

Mobile phase n-hexane: acetone 86:14 (v/v)

Calculation External standard, peak area, linear regression

work by Matyka *et al.* [25] and the official VDLUFA method [8].

chromatography (HPLC) with reversed phase and UV detection.

**Apparatus**: centrifuge, HPLC set, diode array detector.

**Reagents and Solvents:** trypsin 200 FIP – U/g; pepsin 700 FIP – U/g; ammonia; n – hexane; diethyl ether; 99.8% ethyl alcohol; acetone; aluminum oxide, neutral, activity 1; canthaxanthin standard; chloroform. All reagents and solvents should be of analytical grade.

**Apparatus**: ultrasonic bath; vacuum rotary evaporator, HPLC set with Array's diode

The sample is hydrolyzed with an aqueous solution of ammonia at the presence of trypsin and pepsin following extraction with ethyl alcohol and diethyl ether. Purification occurs on the aluminum oxide filled column. The extract prepared in this way should be evaporated, dissolved in the mobile phase, filtered through syringe filters and dosed on the

The procedure of analyzing methionine hydroxy analog was developed on the basis of the

Methionine hydroxy analog is extracted from the sample by 10% acetonitrile, and next hydrolyzed with potassium hydroxide and determined by high performance liquid

**Reagents and Solvents:** acetonitrile; orthophosphoric acid; solution for extraction: acetonitrile - water 10+90 (V/V); solution for hydrolysis: 50% potassium hydroxide (w/v);

Extract methionine hydroxy analog from the feed, with the use of 10% acetonitrile. After centrifuging, perform hydrolysis with potassium hydroxide and next with a solution of orthophosphoric acid. Filter the supernatant through syringe filters and inject on the

### **Special Comment**

