analysis of ractopamine hydrochloride in rawmaterial

AGRICULTURAL MATERIALS

Development and Validation of a Simple Method for RoutineAnalysis of Ractopamine Hydrochloride in Raw Material andFeed Additives by HPLCELLEN FIGUEIREDO FREIRE

University of São Paulo, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto,

Ribeirão Preto, Brazil and Ouro Fino Saúde Animal, Department of Research and Analytical Development, Cravinhos,

Brazil

KEYLLER BASTOS BORGES

University of São Paulo, Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences of Ribeirão Preto,

Ribeirão Preto, Brazil

HÉLIO TANIMOTO, RAQUEL TASSARA NOGUEIRA, and LUCIMARA CRISTIANE TOSO BERTOLINI

Ouro Fino Saúde Animal, Department of Research and Analytical Development, Cravinhos, Brazil

CRISTIANE MASETTO DE GAITANI1

University of São Paulo, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto,

Ribeirão Preto, Brazil

A simple method was optimized and validated for

determination of ractopamine hydrochloride (RAC)

in raw material and feed additives by HPLC for use

in quality control in veterinary industries. The

best-optimized conditions were a C8 column (250 �

4.6 mm id, 5.0 �m particle size) at room temperature

with acetonitrile–100 mM sodium acetate buffer

(pH 5.0; 75 + 25, v/v) mobile phase at a flow rate of

1.0 mL/min and UV detection at 275 nm. With these

conditions, the retention time of RAC was around

5.2 min, and standard curves were linear in the

concentration range of 160–240 �g/mL (correlation

coefficient �0.999). Validation parameters, such as

selectivity, linearity, limit of detection (ranged from

1.60 to 2.05 �g/mL), limit of quantification (ranged

from 4.26 to 6.84 �g/mL), precision (relative

standard deviation �1.87%), accuracy (ranged from

96.97 to 100.54%), and robustness, gave results

within acceptable ranges. Therefore, the developed

method can be successfully applied for the routine

quality control analysis of raw material and

feed additives.

The quantity of drugs used in animal production has

grown exponentially, mainly due to new forms of

intensive livestock. Particularly, the use of

�-adrenergic agonists as growth promoters has grown

considerably in recent years. Among the �-adrenergic

agonists, ractopamine hydrochloride (RAC; Figure 1) is the

most used as a nutrient repartitioning agent by diverting

nutrients from fat deposition in animals to the production of

muscle tissues (1). It promotes reduction of fat, increased

muscle mass, and improved feed utilization efficiency in

swine, cattle, and turkey (2–5). RAC is a phenethanolamine

�-adrenergic agonist that contains 2 chiral carbons, and it is

commercialized as a mixture of 4 stereoisomers in

approximately equal proportions, although studies have

shown that the RR isomer is responsible for a majority of the

leanness-enhancing effects of RAC in rats (6).

RAC was the first compound of this class that received the

approval of the U.S. Food and Drug Administration (FDA) for

use in meat animals as a production enhancer (7). It was

approved by the FDA for use in swine in 2000, under the trade

name of Paylean� (Elanco Animal Health, Greenfield, IN). In

2003, it was approved for use in finishing cattle, under the

trade name of Optaflexx� (Elanco Animal Health). In Brazil,

Ractosuin� (Ouro Fino Saúde Animal, Cravinhos, SP, Brazil)

is produced for use in finishing swine. Although the FDA

approved RAC as a pig feed supplement in 2000, many

countries, such as China, Taiwan, and those of the European

Union, have not allowed its use as a repartitioning agent

because RAC-treated animals used in a human diet could

cause adverse health effects (7).

In the literature, there are various studies concerning the

determination of RAC in various matrixes using different

techniques. Determination of RAC in biological fluids, feeds,

and animal tissues by HPLC (8) with electrochemical (9, 10)

and fluorescence detection (11–15), and by HPLC/MS/MS

(13, 16–19) have been described. GC/MS has also been used

for detection and quantification of RAC (20, 21). In addition,

capillary electrophoresis was used to separate the 4

stereoisomers of RAC (22).

FREIRE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 3, 2009 757

Received November 24, 2008. Accepted by EB December 18, 2008.1Author to whom correspondence should be addressed; e-mail:

[email protected]

Although the analyses of RAC in plasma, serum, urine,

tissues, and liver have already been described in literature, no

method has been reported, to the best of our knowledge, for

the determination of RAC in raw material and feed additives

for routine analysis by HPLC. Whereas the analysis of RAC at

all stages of production is important for safety of all

consumers of meat from animals treated with RAC, the

development of methodologies for simple and rapid analysis

of RAC in raw material and feed additives is required.

Therefore, the purpose of the present study was to develop a

simple, rapid, and efficient method for analysis of RAC in raw

material and feed additives by HPLC with UV absorbance

detection (HPLC-UV).

Experimental

Samples

RAC pure substance was obtained from Riedel de Häen

(Seelze, Germany) with an assigned purity of 96.5%. RAC

(100.5%, raw material) was obtained from Iffect (Shenzhen,

China). Ractosuin (2.05% RAC) and its placebo were

generously donated by Ouro Fino Saúde Animal.

Solvents and Reagents

HPLC grade acetonitrile, glacial acetic acid, and sodium

acetate trihydrate were purchased from J.T. Baker

(Phillipsburg, NJ). All other chemicals were of analytical

grade with the highest purity available. Water was distilled

and purified using a Millipore Milli-Q Plus system

(Bedford, MA).

Instrumentation

The Shimadzu Corp. (Kyoto, Japan) chromatographic

system used to develop and validate this method consisted of

an LC-20 AT pump, SPD-20 A UV-Vis detector, system

controller CBM-20 A, SIL-20 AC automatic injector, CTO-20

A column oven, and DGU-20 A5 degasser. Solution�

software (Shimadzu) was used to control the HPLC system

and for data acquisition. Separation was performed on a C8

column (25 � 4.6 mm id, 5 �m particle size) from Waters

Corp. (Milford, MA). A Security Guard Cartridge System

(Phenomenex, Torrance, CA) C8 guard column (4.0 � 3.0 mm

id, 5 �m) was also used. In addition, a Hitachi UV-Vis NIR�

Model U-3501 spectrometer with 1.0 cm optical path quartz

cuvet was used to determine the best wavelength for

HPLC detection.

Analytical Conditions

The mobile phase consisted of the mixture of acetonitrile

and 100 mM sodium acetate buffer (pH 5.0; 75 + 25, v/v). The

buffer was filtered through a 0.45 �m Millipore membrane

filter, and the mobile phase was filtered and degassed prior to

use. UV detection was at 275 nm. All chromatographic

procedures were conducted at 25�C. A flow rate of

1.0 mL/min was used, and the injection volume was 10 �L for

standards and samples.

Preparation of Reference Solutions

Stock standard solutions of RAC were prepared by

dissolution of the drug in mobile phase to obtain a

concentration of 2.5 mg/mL. This standard solution was

diluted to give the following concentrations: 160, 180, 200,

220, and 240 �g/mL of active pharmaceutical ingredient. All

of these solutions were stored at –20�C in the absence of light.

Working standard solutions were prepared daily by diluting

the stock solutions to an appropriate concentration with the

mobile phase.

Preparation of Sample Solutions

RAC raw material was prepared by dissolution of 62.5 mg

of drug in a 25 mL volumetric flask containing 15 mL of

mobile phase. This solution was sonicated for 2 min, mixed on

a Vortex mixer for 2 min, and diluted to volume with mobile

phase; 800 �L of this solution was then transferred into a

10 mL volumetric flask, diluted to volume with mobile phase,

and filtered through a 0.45 �m membrane filter. This final

solution was injected into the HPLC system.

758 FREIRE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 92, NO. 3, 2009

Figure 1. Chemical structure of ractopamine (RAC).

Table 1. HPLC conditions for the determination of RAC

in raw material and feed additives

Condition Optimized method

Apparatus HPLC Shimadzu

Mobile phase Acetonitrile–100 mM sodium acetate buffer

(pH 5.0; 75 + 25, v/v)

Column C8 Waters Corp.

(25 mm � 4.6 mm id, 5 �m particle size)

Guard column Security Guard Cartridge System C8

(4.0 mm � 3.0 mm id, 5 �m)

Wavelength 275 nm

Flow rate 1 mL/min

Injection volume 10 �L

Temperature 25�C

Retention time 5.2 min

To prepare the placebo spiked with RAC or Ractosuin

samples, the product was ground to a uniform powder, then

975.7 mg was accurately weighed. This amount was

transferred into a 100 mL volumetric flask, diluted with 80 mL

of mobile phase, mixed on a Vortex mixer for 2 min, sonicated

for 15 min, and diluted to volume with mobile phase. This

solution was filtered through a 0.45 �m membrane filter and

injected into the HPLC system.

Validation of the Method

The method was validated for analysis of raw material and

Ractosuin samples. Validation for raw material was realized

using standard solutions of RAC in the mobile phase, whereas

validation for the analysis of Ractosuin was performed in

placebo spiked with RAC standard solutions. The stability test

of standard solutions and system suitability were performed

before starting the validation. The following parameters were

studied: selectivity, linearity, range, precision, accuracy, limit

of detection (LOD), limit of quantification (LOQ), and

robustness, following the U.S. Pharmacopeia (USP)

guidelines (23).

(a) Standard solution stability.—The stability of the test

solution prepared in mobile phase was evaluated. A solution

of 200 �g/mL RAC was stored at room temperature

(autosampler) and analyzed at intervals of 6, 13, 18, and 27 h.

The responses for the aged solution were evaluated against a

freshly prepared standard solution.

(b) System suitability.—The system suitability test was

also performed to evaluate the resolution and reproducibility

of the system for the analysis to be performed, using

5 replicate injections of a standard solution containing

200 �g/mL RAC.

(c) Selectivity.—To evaluate the selectivity of the method,

the placebo samples (excipients) were analyzed and compared

with the RAC standard solution (200 �g/mL) and Ractosuin

samples. Therefore, the selectivity of the method toward the

drug was established by checking the interference of placebo

peaks in chromatograms of RAC samples. The peak areas and

retention times were utilized.

(d) Linearity and range.—Linearity test solutions for the

assay method were determined by constructing 3 standard

curves prepared at 5 concentration levels from 80 to 120% of

the assay analyte concentration. Standard concentrations of

RAC in the range of 160–240 �g/mL (160, 180, 200, 220, and

240 �g/mL) were prepared in the mobile phase. Three

replicate 10 �L injections were made of the standard solution

to verify repeatability of the detector response at each

concentration. The peak areas of the chromatograms were

plotted against the concentrations of RAC to obtain the

respective standard curves. The 5 concentrations of the

standard solutions were subjected to regression analysis by

the least-squares method to calculate the calibration equation

and correlation coefficient (r).

(e) Limits of detection and quantification.—The LOD and

LOQ values were directly calculated by using the calibration

curve. The LOD and LOQ were calculated from the slope and

the standard deviation (SD) of the intercept of the mean of

3 standard curves, determined by a linear regression model, as

defined by the International Conference on

Harmonization (24). The factors 3.3 and 10.0 for LOD and

LOQ, respectively, were multiplied by the ratio from the

residual SD and the slope (corresponding to the standard error

of the slope).


Table 2. Test of standard solution stability

Found, %

Time, h Raw materiala Feed additivea

6 99.90 99.95

13 99.65 99.77

18 99.47 99.50

27 99.30 99.39

RSD, % 0.29 0.27

a Mean of 3 replicate analyses.

Figure 2. Typical chromatogram of RAC in (A) rawmaterial and (B) feed additive. The spectra of UV-Visabsorbance of RAC are shown in upper right of figures.

(f) Precision and accuracy.—The precision of the method

was determined by repeatability and intermediate precision

studies. Repeatability was examined by 3 evaluations at

3 different RAC sample concentrations (160, 200, and

240 �g/mL) on the same day under the same experimental

conditions. The intermediate precision of the method was

assessed by performing the analysis on 2 different days

(interday), and by other analysts performing the analysis in the

same laboratory (between analysts, 2 analysts). The accuracy

was evaluated by applying the proposed method to the analysis

of an in-house mixture of the placebo with known amounts of

RAC to obtain concentrations of 160, 200, and 240 �g/mL,

equivalent to 80, 100, and 120% of the label values. The

accuracy was calculated as the percentage of the drug recovered

from the formulation matrix. These studies were performed for

raw material and placebo spiked with RAC.

(g) Robustness.—The robustness was determined for raw

material and placebo spiked with RAC by analyzing the same

samples under a variety of conditions of the method

parameters, such as flow rate, column temperature, and

different columns.

Results and Discussion

HPLC Method Development

The development and validation of a simple and suitable

HPLC method for the quantitative determination of RAC in

raw material and feed additives was performed. The analytical

conditions were selected after testing different parameters


Table 3. Results of system suitability test for RAC determination

ParameterUSP

recommendationDay

determinationa Minimum Maximum RSD, %b Status

Retention time, min RSD, % <2 Day 1 5.189 5.194 0.04 Approved

Day 2 5.162 5.165 0.03 Approved

Repeatability RSD, % <2 Day 1 856222 860238 0.18 Approved

Day 2 857664 859278 0.07 Approved

Theoretical plates >2000 Day 1 6292.06 6431.98 0.81 Approved

Day 2 6450.14 6508.88 0.38 Approved

Asymmetry <2 Day 1 1.165 1.169 0.12 Approved

Day 2 1.162 1.167 0.14 Approved

a On the first day the analyses were made by analyst 1, and on the second day by analyst 2.b Values from 5 replicates.

Figure 3. Representative chromatogram of (A) mobile phase and (B) a placebo of Ractosuin.

such as buffer composition, buffer concentration, mobile

phase composition, and other chromatographic conditions.

The proportions of the organic and aqueous phases were

adjusted to obtain a rapid and simple assay method for RAC

with a reasonable run time, suitable retention time, and

sharp peak.

Satisfactory resolution was obtained using the mobile

phase sodium acetate buffer (pH 5.0)–acetonitrile (25 + 75,

v/v) with a flow rate of 1 mL/min. For quantitative analytical

purposes, the detection wavelength was set at 275 nm, which

provided better reproducibility and lower potential for

interference than the other UV bands. Table 1 summarizes the

optimized HPLC conditions of the analytical method. Under

the chosen experimental conditions, the chromatograms of

RAC in raw material (Figure 2A) and feed additives

(Figure 2B) showed a peak around 5.2 min.

Standard Solution Stability

Table 2 shows the results obtained in the stability study of

200 �g/mL RAC solution at different time intervals. The

relative standard deviation (RSD) values of the RAC assay

stability experiments were lower than 0.3%. The data

obtained in the experiments proved that the sample solutions

used during assays were stable up to 27 h.

System Suitability

A system suitability test of the chromatographic system

was performed before each validation run. Five replicate

injections of standard solutions were made, and asymmetry,

theoretical plate number, and RSD of the peak areas and

retention times were determined. For all system suitability

injections, asymmetries were <2.0, theoretical plate numbers

were >5000, and RSD of the peak areas and retention time

was <2.0% (Table 3).


Figure 4. Chromatogram illustrating the separation of RAC and its adjacent interferents: (1) interferent A; (2) RAC;

and (3) interferent B.

Table 4. Chromatographic parameters obtained for the

RAC determination in raw material and feed additive

Parameter Day Interferent A RAC Interferent B

Rsa

1 2.23 6.01

2 2.20 6.06

kb

1 0.92 1.14 1.77

2 0.92 1.14 1.75

�c

1 1.24 1.55

2 1.24 1.54

Nd

1 7612.46 6349.98 11 460.89

2 7378.42 6481.91 11 749.28

a Rs = Resolution.b k = Retention factor for the first peak eluted (tm = 2.43 min, defined

as the first significant baseline disturbance, corresponding to theretention time of a nonretained solute).

c� = Separation factor.

d N = Theoretical plates.

Selectivity

The selectivity of analytical method for RAC is presented

in Figure 3. The chromatograms indicate that the developed

method was successful in separating the drug and placebo

products. The peak areas obtained for raw material and feed

additives (n = 3, for 200 �g/mL RAC standard solution and

Ractosuin samples) were compared. The RSD value observed

was 0.74%. The retention times had good reproducibility, with

an RSD value of 0.05%. Figure 4 presents the chromatogram

of RAC and its adjacent interferents. In addition, Table 4

shows the chromatographic parameters obtained for RAC and

the adjacent interferents.

Linearity and Range

The calibration curve was prepared by plotting the peak

area of RAC against drug concentration; it was linear in the

range of 160–240 �g/mL. Peak area and concentration were

subjected to least-squares linear regression analysis to

calculate the calibration equation and r value. The linear

equations, correlation coefficient, and RSD values are


Table 5. Linearity and limits of detection and quantification of the methods for RAC in raw material and feed

additives

Parameter Day Raw material Feed additive

Linear equationa

1 y = 4307.3x – 6397.0 y = 4305.8x – 7413.2

2 y = 4258.2x + 2579.2 y = 4253.4x + 2820.9

Correlation coefficient (r) 1 0.9999 0.9999

2 0.9999 0.9999

Range, �g/mL 1 160–240 160–240

2 160–240 160–240

RSD, %b

1 0.38 0.39

2 0.84 0.84

LOD, �g/mL 1 1.60 1.28

2 1.99 2.05

LOQ, �g/mL 1 5.33 4.26

2 6.63 6.84

a Calibration curves were determined in triplicate (n = 3) for concentrations of 160, 180, 200, 220, and 240 �g/mL; y = Ax + B, where y is the

peak area of RAC, A is the slope, B is the intercept, and x is the concentration of the measured solution in �g/mL.b RSD = Relative standard deviation of the slope of the calibration curve.

Table 6. Precision and accuracy of the method for analysis of RAC in raw material and feed additives

Nominal concentration, �g/mL

Raw material Feed additive

Parameter Day 160.00 200.00 240.00 160.00 200.00 240.00

Within day (n = 3)

Analyzed concentration, �g/mL 1 160.28 200.59 240.31 156.42 194.30 236.46

2 160.31 201.07 239.83 156.57 193.93 236.34

Precision (RSD), % 1 0.26 0.35 0.05 0.94 0.47 0.07

2 0.35 1.09 0.09 1.87 0.64 0.03

Accuracy, % 1 100.18 100.30 100.13 97.76 97.15 98.52

2 100.19 100.54 99.93 97.86 96.97 98.48

Between day (n = 2)

Analyzed concentration, �g/mL 160.30 200.83 243.07 156.49 194.12 236.40

Precision (RSD), % 0.25 0.75 0.13 1.33 0.51 0.06

Accuracy, % 100.19 100.42 100.03 97.81 97.06 98.50

presented in Table 5. All r values were �0.9999, showing

excellent linearity.

LOD and LOQ

For the calculation of the LOD and LOQ, the calibration

equations for RAC were generated by using the mean values

of the 3 independent standard curves. The values calculated

for the LOD and LOQ are shown in Table 5.

Precision and Accuracy

The precision and accuracy of the method were evaluated

by calculating the RSD for 3 determinations of RAC solution

at 3 concentrations (160, 200, and 240 �g/mL) performed on

2 days under the same experimental conditions. Table 6 shows

within-day (n = 3 for each concentration) and between-day

(n = 2, on 2 different days and 2 analysts) assays of RAC raw

material and placebo spiked with RAC. These results show

that the method is accurate within the desired range.

Robustness

The results of the robustness study of the developed assay

method are given in Table 7. During all variation conditions,

the assay value of the test preparation solution was not

affected, and it was in accordance with the actual value.

System suitability parameters were also found to be

satisfactory; hence, the analytical method can be considered to

be robust.

Application of the Method

The applicability of the proposed method was shown by

the determination of RAC in raw material and Ractosuin

product. The results obtained were satisfactorily accurate and

precise, as indicated by the excellent recovery and RSD values

(Table 8). Placebo of Ractosuin did not interfere with the

assay. The raw material and Ractosuin product were in

accordance with their specifications.

Conclusions

A rapid and reliable isocratic HPLC-UV method for

determination of RAC was developed and validated to be

routinely applied to analysis of raw material and

feed additives. The developed procedure was proven to

be selective, linear, precise, accurate, robust, and

stability-indicating. In addition, its chromatographic

run time of 5.2 min allows the analysis of a large number

of samples in a short period of time. Therefore, this HPLC-UV

method can be used for routine quality control analysis and

stability tests.


Table 7. Chromatographic conditions and range investigated during robustness testing

RAC

Raw materiala Feed additivea

Variable Range investigated Concn, �g/mLb Found, %a Concn, �g/mLb Found, %a

Flow rate, mL/min 0.98 203.54 101.77 198.25 99.13

1.00 200.49 100.25 199.64 99.82

1.02 197.72 98.86 197.93 98.97

Column C8 column B 201.43 100.72 200.67 100.34

C8 column A 200.49 100.25 199.64 99.82

C8 column C 201.56 100.78 201.03 100.52

Column temperature, �C 24 201.37 100.69 200.21 100.11

25 200.49 100.25 199.64 99.82

26 201.41 100.71 201.27 100.64

a Mean of 3 replicate analyses.b Concn = Concentration.

Table 8. Determination of RAC in raw material and feed

additive samples

Sample

Raw material(100.5%)a

Feed additive(Ractosuin 2.05%)a

Found, %b RSD, % Found, %b RSD, %

1 99.72 97.66

2 99.28 0.66 96.98 0.47

3 99.23 97.12

4 100.66 97.95

a Product specification.b Mean of 3 replicate analyses.

Acknowledgments

We are grateful to Ouro Fino Saúde Animal for providing

the RAC reference substance, Ractosuin, and its placebo. We

are also grateful to Fundação de Amparo à Pesquisa

do Estado de São Paulo, Conselho Nacional de

Desenvolvimento Científico e Tecnológico, and Coordenação

de Aperfeiçoamento de Pessoal de Nível Superior for

financial support and for granting research fellowships.

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analysis of ractopamine hydrochloride in rawmaterial

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