AGRICULTURAL MATERIALS

Liquid Chromatographic Determination of Monensin in Premix

and Animal Feeds: Collaborative Study
COLEMAN ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 80, NO. 4, 1997
MARK R. COLEMAN, JOHN W. MORAN, and DANIEL H. MOWREY
Elanco Animal Health, Eli Lilly and Company, Indianapolis, IN 46140
Collaborators: J.P. Antalick; D.J. Bark; D.A. Bridges; N.L. Britton; H.M. Campbell; J. Cardenas; F.H. Johannsen; V.B. Reeves;
A. Siirila; R.L. Smallidge; L. Wetzler

An interlaboratory study of a liquid chromatographic (LC) method for determining monensin

in premix (6080 g/lb or 132176 mg/g) and animal
feeds (5200 g/ton or 0.00550.22 mg/g) was conducted in laboratoriesin the United States, Canada,
France, and Germany. The LC system used a reversed-phase column, postcolumn derivatization
with vanillin, and UV detection. The method separates monensin from other ionophores such as
narasin and salinomycin. Each laboratory analyzed
a total of 20 samples of premix, liquid feed supplements, poultry, and cattle feeds. Concentrations of
monensin in all samples ranged from 0 to 176 mg/g
(80 g/lb). Reproducibility relative standard devia%. For
tion (RSDR) for premix ranged from 2.8 to 3.4%
feed samples containing monensin, repeatability
standard deviation (sr) ranged from 0.9 to 7.0. Reproducibility standard deviation (sR) ranged from
1.2 to 11. Repeatability relative standard deviation
% and RSDR values(RSDr) ranged from 6.1 to 21%
ranged from 8.6 to 25%
%. Sample preparation for the
LC method is less labor intensive than that for the
microbiological assays. The LC assay is more efficient than the microbiological assays. This LC
method for determination of monensin in premix and
animal feeds has been adopted first action by AOAC
INTERNATIONAL.

poultry. The approved concentrations of monensin in various

matrixes may range from 0.0055 mg/g (about 5 g/ton) to
200 g/kg. The limit of quantitation for this method is 4 g/g
(0.004 mg/g).
Collaborative Study

Collaborators
Ten laboratories in the United States, Canada, France, and
Germany participated and completed the collaborative study.
Each potential collaborator received a letter of invitation, a
copy of the method, a questionnaire, and an outline of the collaborative study. The letter of invitation explained the purpose
of the study and provided information for the potential collaborator to review. Potential collaborators that returned the questionnaire were asked to analyze 2 familiarization samples and
a resolution mixture to demonstrate that their chromatographic
system was suitable for this study. Each collaborator received
a letter explaining the need for familiarization samples, instructions for performing analyses, and data forms for LC control
parameters and samples. Laboratories that successfully completed analyses of familiarization samples and the resolution
mixture were asked to participate in the final study. Each collaborator received a letter requesting participation, instructions for
performing analyses, and data forms for LC control parameters
and samples.

Preparation of Test Samples

he AOAC methods for determining monensin in feeds
are microbiological (1). Both plate and turbidimetric
methods are approved. A liquid chromatographic (LC)
method was developed and validated for quantitative determination of monensin in bulk drug, premix, and animal feeds (2,
3). Monensin is marketed as Rumensin for cattle and Coban for

Submitted for publication September 23, 1996.

The recommendation was approved by the Methods Committee on
Feeds, Fertilizer, and Related Agricultural Topics, and was adopted by the
Official Methods Board of the Association. See Official Methods Board
Actions (1997) J. AOAC Int. 80, 35A, and Official Methods Board
Actions (1997) Inside Laboratory Management, March issue.

A protocol was written and approved according to the guidelines outlined by AOAC INTERNATIONAL (4). All collaborators received monensin reference standard from the Associate
Referee. In addition, each collaborator received narasin reference standard to be used for the resolution mixture and a series
of samples containing monensin (Table 1). Youdens matched
pairs covering the concentration range described were used in
this study. In addition to solid and liquid feed samples, each
collaborator also received samples of Coban 60 premix (containing 60 g monensin activity per pound, or 132 mg/g) and
Rumensin 80 (80 g monensin activity per pound, or 176 mg/g).
A poultry ration (Tables 2 and 3) was medicated with Coban 60
premix to contain 30 and 110 g monensin activity per ton

(0.033 and 0.121 mg/g). Two cattle rations (Tables 47) were
used to prepare samples in the 510 g/ton (0.00550.011 mg/g)
concentration range, and one cattle ration (Tables 6 and 7) was
used to prepare a cattle ration at 30 g monensin activity per ton.
Rumensin 80 premix was used to medicate liquid feed supplements to contain 25 and 200 g monensin activity per ton (0.0275
0.22 mg/g). Each collaborator received blind nonmedicated samples of each of the rations. All samples were preweighed into
extraction containers and sent to each laboratory.
997.04 Monensin in Premix and Animal Feeds,
Liquid Chromatographic Method
First Action 1997
(Suitable for determination of monensin in range from
0.0055 to 200 g/kg in all types of premixes and animal feeds.)
Caution: See Appendix B: safety notes on safe handling of
acidssulfuric acid. See material safety data sheets, or equivalent, for each reagent. Dispose of waste solvents in an appropriate manner compatible with applicable environmental rules and
regulations.
Method Performance:
See Table 997.04 for method performance data.

A. Principle
Monensin is extracted from premixes and feeds with methanolwater (90 + 10, v/v) and analyzed by liquid chromatography (LC) with postcolumn derivatization. Acid reaction of
monensin with vanillin is measured by variable-wavelength
detector operating at 520 nm. There is no interference from
narasin, salinomycin, and lasalocid.

B. Apparatus
(a) Liquid chromatograph.Equipped with recorder or integrator (manual or computer), pulse-dampened LC pump [for
LC mobile phase, C(g)] and reagent pump [for vanillin reagent,
C(h)] both operating at 0.7 mL/min, variable-wavelength absorbance detector operating at 520 nm with rise time of 1.0 and
range of 0.2 (or as needed); 90 tee positioned in such way so
inlet flows directly oppose one another; and stainless steel coil
(6.1 m 0.51 mm id) enclosed in 98.0C heater. (Comparable
components may be substituted. Parameters listed may be
modified to compensate for daily variations in instrument performance.) Operating conditions: injection volume, 200 L;
column temperature, ambient; reaction coil temperature, 98C;
flow rate (both pumps), 0.7 mL/min. Approximate retention
times: monensin A, 640 s; monensin B, 560 s; monensin C/D,
730 s (see Figure 997.04A for chromatogram of typical system
suitability sample, Figure 997.04B for chromatogram of rumensin medicated article, and Figure 997.04C for chromatogram of monensin medicated feed.)
Note: When system is not in use for 23 days, or at regular
intervals during use periods, flush system with methanol to prolong life of components.

(b) LC column.250 4.6 mm id (Whatman Partisil 5

ODS-3 25, or equivalent). C18 guard column may be used to
extend life of column.
(c) LC vials.
(d) Mechanical shaker.Wrist action shaker, Gyratory
shaker or equivalent.
(e) Filter.0.450.5 m porosity; Teflon or chemical
resistant.
(f) Containers.5001000 mL, glass or polypropylene.
(g) Volumetric flasks.100 and 200 mL.
(h) Magnetic stirrer.
(i) Teflon-coated stir bars.
(j) Feed grinder.Centrifugal grinding mill equipped
with 3 mm screen, or equivalent.
(k) Feed mixer.
(l) Balances.(1) Top loading and (2) analytical.

C. Reagents
(a) Solvent.Methanol, LC grade.
(b) Purified H2O.LC grade.
(c) Sulfuric acid.Analytical grade.
(d) Glacial acetic acid.ACS grade.
(e) Vanillin.99% purity.
(f) Neutralized methanol.Add 1 g sodium bicarbonate to
4 L methanol. Mix well and, if needed, filter through 11 m
filter paper.
(g) LC mobile phase.MethanolH2Oglacial acetic acid
solution (94 + 6 + 0.1, v/v/v). Degas prior to use.
(h) Vanillin reagent.Methanolsulfuric acidvanillin solution (95 + 2 + 3, v/v/w). While stirring gently, add slowly and
carefully 20 mL concentrated sulfuric acid to 950 mL methanol, C(a). (Caution: Avoid splattering when adding sulfuric
acid to methanol. Add acid slowly and carefully, using pipet or
other suitable equipment. Do not add sulfuric acid to methanol
by pouring from beaker or graduated cylinder.) Allow methanolsulfuric acid solution to cool to room temperature. Add
30.0 g vanillin while stirring gently. Vanillin reagent is stable
up to 4 weeks when stored at room temperature in dark amber
or foil-covered container.
(i) Extraction solution.MethanolH2O solution (90 + 10, v/v).
(j) Monensin standard solutions.(1) Monensin standard
stock solution.1 mg/mL. Accurately weigh 100 mg monensin reference standard (available from Eli Lilly and Company,
Lilly Corporate Center, Indianapolis, IN 46285) into 100 mL
volumetric flask and dilute to volume with methanol, C(a). (2)
Monensin working standard solutions.Quantitatively dilute
monensin standard stock solution with methanol to obtain
monensin concentrations of 1.0, 5.0, 20.0, and 40.0 g/mL.
(Note: When analyzing premix samples use 5.0, 20.0, and
40.0 g/mL monensin standard working solutions.) Monensin
standard solutions are stable up to 4 weeks when stored in refrigerator or at room temperature protected from direct sunlight.
(k) Resolution solution.Containing 540 g monensin/mL and 540 g narasin/mL. Resolution solution can be
prepared as follows: Accurately weigh ca 100 mg monensin
sodium reference standard and 300 mg narasin reference standard into 100 mL volumetric flask. Dilute to volume with neu-

tralized methanol, C(f). Mix well. From this solution pipet

2 mL into 200 mL volumetric flask and dilute to volume with
extraction solution, C(i). Mix well. Resolution solution is stable
up to 2 weeks when stored in refrigerator or at room temperature protected from direct sunlight.

D. Preparation of Test Sample

(a) Premix.Accurately weigh ca 5 g premix into 500 mL
jar or other suitable container. Add 200 2 mL extraction solution, C(i), cap, and mix thoroughly 1 h on mechanical shaker.
Allow sample solids to settle. Based on theoretical monensin
concentration, dilute sample to obtain final monensin concentration within standard curve levels. Filter aliquot of extract and
proceed to LC determination, F.
(b) Feed.Grind feed sample with grinder, B(j) ,and then
mix entire sample 10 min using mixer, B(k). Accurately weigh
ca 50 g sample into 500 mL jar or other suitable container. Proceed as in (a) starting with Add 200 2 mL extraction solution ...
(c) Liquid feed supplements.Note: Settling of insoluble
matter in fortified liquid feed supplements presents sampling
problem. Ensure adequate mixing of liquid feed supplements
prior to sampling. Thoroughly mix samples by agitation with
gyratory shaker, propeller mixer, magnetic stir bar, or equivalent apparatus depending on viscosity of sample. While continuing to mix, rapidly (to prevent settling) transfer ca 40 g to
200 mL volumetric flask. Add 100150 mL extraction solution, C(i), cap, and mix thoroughly. Dilute to volume with extraction solution. Allow mixture to settle. Dilute aliquot of extract with extraction solution to obtain final monensin
concentration of ca 20 g/mL. Filter aliquot of diluted extract
through 0.45 m filter, B(e), and proceed to LC determination, F.

E. System Suitability
(a) Resolution.Inject resolution solution onto LC column. Calculate resolution factor, Rs, for each pair of adjacent
peaks as follows (see Figure 997.04A):
t2 t1
Rs =
(tw1 + tw2) 0.5
where t1, t2, ..., tn = retention volume of peak maximum, cm;
tw1, tw2, ..., twn = triangulated peak width at baseline, cm.
Example: t1 = 10.65 cm; t2 = 11.85 cm; t3 = 15.55 cm; tw1
= 0.85 cm; tw2 = 0.95 cm; tw3 = 1.00 cm.
11.85 10.65
(0.85 + 0.95) 0.5
= 1.33 (must be 1.25)

Monensin B Monensin A, Rs =

If resolution does not meet requirements specified adjacent

to calculations, adjust LC conditions to improve resolution and
reinject resolution solution onto LC system.
(b) Tailing factor.Tailing factor, Tf, for monensin factor
A of reference standards must be <1.4. Calculate Tf as follows:
Tf =

10% width
(retention time time of front 10% point) 2

where retention time = time of the maximum of the fitted

Gaussian curve; 10% width = (time of back 10% point time
of front 10% point). A 10% point is where the response of a side
of the peak reaches a height equal to 10% of the peak maximum.
(c) Linearity of standard curve.The correlation coefficient, R2, for standard curve must be 0.995.
If any of the above parameters does not meet system suitability, adjust mobile phase. After adjustment, parameters must
meet system suitability prior to sample analysis.
(d) Limit of quantitation.4 g/g (0.004 mg/g).

F. LC Determination
Note: The most critical parameters in LC measurement system are H2O content, reactor temperature, sulfuric acidvanillin concentrations, and flow rates.
Before analyzing test sample, ensure that LC system meets
system suitability parameters by injecting resolution solution,
C(k). Then proceed by injecting 200 L standard solutions followed by analytical samples.
Measure peak area response, PR, at retention volume of
monensin factor A and monensin factor B for each sample.

G. Calculations
Using measured responses, construct linear regression plot
of standard curve of monensin factor A to determine concentration of monensin factors A and B in each samples. Determine
biopotency for each factor as follows:
Biopotency = g/mL (from standard curve)
V
BCF f

Ws

where V = extraction volume, mL (e.g., 200 mL); Ws = weight

of sample, g; f = dilution factor; BCF = biopotency conversion
factor [BCF (Factor A) = 1.000; BCF (Factor B) = 0.280].
Take the sum of individual biopotency values for monensin
A and monensin B to obtain total biopotency for monensin.
(Note: Refer to the Reference Standard Profile [available from
Eli Lilly and Company] for percent Factor A content of the
current Reference Standard.)
Ref.: J. AOAC Int. 80, 693702(1997)
Results and Discussion

Monensin A Narasin A, Rs =

15.55 11.85
(0.95 + 1.00) 0.5
= 3.79 (must be 3.50)

All 10 laboratories that agreed to participate in the collaborative study completed analyses and submitted data to the Associate Referee.

System Suitability
Each collaborator was asked to determine several system
suitability parameters prior to analysis of samples. System suitability parameters used by the sponsor laboratory were provided as targets for the collaborator. In addition, acceptable parameters were listed and the collaborators were to contact the
Associate Referee if these parameters could not be met. The
key parameters were resolution factors (Rs), retention time, and
tailing. All laboratories met the criteria for resolution factors
and tailing as indicated in Table 8. The method indicates that
the tailing factor for monensin A and narasin should be less than
1.4. Laboratories were also asked to calculate tailing for
monensin factor B. The tailing factor for monensin B was less
than 1.4 for all laboratories, except for laboratory F, which had
a tailing factor of 1.48. Among the system suitability parameters, retention time was the most difficult parameter to determine. Ranges were provided for both monensin A and narasin.
Three laboratories did not meet the 600720 s window for
monensin A and 2 laboratories did not meet the 7501000 s
window for narasin. Several collaborators indicated that this
parameter should be changed on the basis of the findings of the
collaborative study. Several collaborators indicated that the retention time window should be provided as a guide but that it
should not be used as a system suitability parameter because the
retention time may shift with new columns and/or mobile
phase.

Nonmedicated Control Feeds

Each collaborator received nonmedicated portions of each
of the rations (poultry ration, 2 cattle rations, and liquid feed
supplement), which were provided as blind samples. A summary of the results is listed in Table 9. All results were rounded
to one place past the decimal point. It was anticipated that each
laboratory would report results as below the limit of the standard curve concentrations or <4 ppm. A sample containing
<4 ppm monensin would not have a monensin peak greater
than or equal to the 1 g/mL standard, and with a dilution factor
of 4 (50 g/200 mL) the result would be reported as <4 ppm
monensin. Although each laboratory chose to report these results differently, only laboratory A for cattle feed 2 observed a
peak at the retention time window of monensin.

Premix Samples
Each collaborator received samples of Coban 60 and Rumensin 80 premix. Results from the 10 laboratories are listed
in Table 10. Statistical analysis suggested that results from
laboratories C and E are outliers. Table 10 lists results from all
10 laboratories and the outliers. Two samples in laboratory E
may have been reversed, but no explanation could be given for
the low results obtained in laboratory C.

Feed Samples
Results for the poultry, cattle, and liquid feed samples are
given in Table 11. All laboratories completed these analyses.
The overall mean and RSD for each sample type by concentration are also included in Table 11.

Statistical Analysis of Data

Data were analyzed according to AOAC INTERNATIONAL Guidelines for Collaborative Study Procedures and
AOAC INTERNATIONAL statistical software (4, 5). Because
collaborators received only one premix sample at_each concentration, estimates were calculated for the mean (x), reproducibility (R), reproducibility standard deviation (sR), and reproducibility relative standard deviation (RSDR). Premix data
were checked for outliers (P = 0.01) with the Cochran and
Grubbs procedures outlined in AOAC INTERNATIONAL
guidelines (4, 5). For feed samples, a Youden matched pair
analysis
_ was conducted to yield estimates of precision parameters: x, R, sR, RSDR, repeatability (r), repeatability standard deviation (sr), and repeatability relative standard deviation
(RSDr). Feed samples were checked for outliers by using the
Cochran and Grubbs procedures outlined in AOAC INTERNATIONAL guidelines (4, 5). If outliers were detected, data
for that particular material were deleted and calculations rerun.
For each material, no more than 2of 9 values were were deleted.

Statistical Summary of Premix Data

Statistical summary for the Coban and Rumensin premix
data is presented in Table 997.04. RSDR values were 2.8% for
Coban premix and 3.4% for Rumensin premix.

Statistical Summary of Feed Data

The statistical summary for each feed type by concentration
is presented in Table 997.04. The sr values ranged from 0.9 to
7.0; sR values ranged from 1.2 to 11. RSDr values ranged from
6.1 to 21%, and RSDR values ranged from 8.6 to 25%. Data for
liquid feed supplements containing monensin at 200 g/ton
ranged from 59.0 to 22 639.0 g/ton and were too variable for
statistical analyses.

Collaborators Comments
Collaborators found no major problems with the procedure
and provided the following comments: (1) sample containers
leaked solvent when placed on end, (2) liquid feed supplements
were difficult to extract, (3) vanillin reagent was corrosive, (4)
vanillin reagent pump required frequent repair, (5) calculation
section of the method needed to be modified to improve clarity,
and (6) retention time window should be provided as a guide
and not used as a system suitability parameter because the retention time may shift with new columns and/or mobile phase.
After evaluating the comments received from collaborators,
the following actions were taken: (1) Alternative sample extraction containers currently are being evaluated, although the
Associate Referee did not intend for the extraction containers
to be placed on their sides. (2) Many problems associated with
liquid feed supplements resulted fromattempts by collaborators
to quantitatively transfer samples from the supplied container
to an alternative container. The Associate Referee did not intend for samples to be transferred. In everyday practice, the
analyst will sample a liquid feed supplement while it is being
mixed and transfer the subsample directly to the extraction vessel. (3) The vanillin reagent is corrosive because of the presence

of sulfuric acid. This acid is needed for the reaction, and problems can be reduced by avoiding contact with this reagent. (4)
The vanillin reagent pump should not require frequent repair if
it is suitable for use with a reagent containing sulfuric acid and
is flushed as recommended. (5) The calculation section has
been clarified. (6) Retention times will be listed as guides in the
method but will not be a system suitability requirement.
The accuracy and precision of the method as determined in
the collaborative study were more variable than had been expected because of the experience and data generated by the
originating laboratory. This observation can be explained in
several ways. First, for many of the laboratories, this was their
first experience with a postcolumn derivatization system. Second, many problems experienced by collaborators during the
collaborative phase of the study have been overcome by minor
method modifications.

Douglas J. Bark, Woodson-Tenent Laboratories, Inc.,

Memphis, TN
Debbie A. Bridges, Eli Lilly and Company, Greenfield, IN
Harold M. Campbell, Agriculture Canada, Ottawa, ON,
Canada
Jose Cardenas, Elanco Analytical Laboratory,
Indianapolis, IN
Robert L. Smallidge and Nancy L. Britton, Office of
Indiana State Chemist, West Lafayette, IN
Friedrich H. Johannsen, Lufa Kiel, W-2300 Kiel 1,
Germany
Valerie B. Reeves, U.S. Food and Drug Administration,
Agricultural Research Center East, Beltsville, MD
Alan Siirila, Hazleton Wisconsin, Madison, WI
LuAnn Wetzler, Nebraska Department of Agriculture
Laboratory, Lincoln, NE

Recommendation

References

On the basis of the results of this study, it is recommended

that the LC method for determining monensin in premix and
animal feeds using postcolumn derivatization be adopted first
action.

(1) Official Methods of Analysis (1995) 16th Ed., AOAC INTERNATIONAL, Arlington, VA
(2) Rodewald, J.M., Moran, J.W., Donoho, A.L., & Coleman,
M.R. (1992) J. AOAC Int. 75, 272279
(3) Coleman, M.R., Macy, T.D., Moran, J.W., & Rodewald, J.M.
(1994) J. AOAC Int. 77, 10651072
(4) AOAC Official Methods Program (1995) Associate Referees
Manual on Development, Study, Review, and Approval Process for AOAC Official Methods, AOAC INTERNATIONAL,
Arlington, VA
(5) Youden, W.J., & Steiner, E.H. (1975) Statistical Manual of
the AOAC, Association of Official Analytical Chemists, Arlington, VA

Acknowledgments
We thank the collaborators and their associates for their cooperation and participation in this study. We thank General
Referee Robert L. Smallidge for his assistance in designing,
planning, and implementing this study.
J.P. Antalick, I.E.E.B., Bordeaux, France

Table 997.04. Method performance for determination of monensin in premixes and animal feed by liquid
chromatography with postcolumn derivatization
Sample
Coban premix
Rumensin premix
Poultry feed
Poultry feed
Cattle feed 1
Cattle feed 2
Cattle feed 2
Liquid feed supplement

Mean
130.2 mg/g
190.3 mg/g
32.4 g/t
114.8 g/t
8.6 g/t
9.0 g/t
32.4 g/t
25.3 g/t

Monensin
132 mg/g
176 mg/g
30 g/t
100 g/t
7 g/t
7 g/t
30 g/t
25 g/t

sr

sR

3.8
7.0
0.9
1.4
2.6
5.3

3.6
6.5
3.8
11
1.2
1.4
2.8
6.4

RSDr, %

RSDR, %

12
6.1
11
15
7.9
21

2.8
3.4
12
9.8
14
15
8.6
25

11
20
2.6
3.8
7.1
15

10
18
11
31
3.2
3.8
7.8
18

Table 1. Samples supplied to each collaborator for

evaluation of the monesin LC assay
Sample
Coban 60 premix
Rumensin 80 premix
Poultry ration, 30 g/ton
Poultry ration, 110 g/ton
Cattle ration No. 1, 7 g/ton
Cattle ration No. 2, 7 g/ton
Cattle ration, 30 g/ton
LFS, 25 g/ton
LFS, 200 g/ton
Control poultry ration
Control cattle ration 1
Control cattle ration 2
Control LFS

No. of Youden
matched pairs

1
1
1
1
1
1
1

No. of
samples
1
1
2
2
2
2
2
2
2
1
1
1
1

Table 2. Composition of poultry ration (broiler starter)

Ingredient
Ground yellow corn
Animal, vegetable fat
Soybean meal, 48%
Fish meal, Menhaden
Feather meal, hydrate
Dicalcium phosphate
Ground limestone
Salt
a
Vitamin premix TK-01 (1.03)
b
Trace mineral premix TK-01 (1.02)
Methionine hydroxy analog
Total
a

Amount, %

Amount, lbs/ton

56.04
3.13
32.37
2.50
2.50
1.66
0.77
0.30
0.50
0.10
0.13

1120.80
62.60
647.40
50.00
50.00
33.20
15.40
6.00
10.00
2.00
2.60

100.00

2000.00

Vitamin premix provides 3000 IU vitamin A, 900 ICU vitamin D3, 40 mg vitamin E, 0.7 mg vitamin K, 1000 mg choline, 70 mg niacin, 4 mg
pantothenic acid, 4 mg riboflavin, 100 g vitamin B12, 100 g biotin, and 125 mg ethoxyquin/kg complete feed.
Trace mineral premix provides 75 mg manganese, 50 mg zinc, 25 mg iron, and 1 mg iodine/kg complete feed.

Table 3. Calculated analysis for poultry ration (broiler starter)

Component
Crude protein, %
Crude fat, %
Kcal ME/kg
ME/CP ratio
Fiber, %
Ash, %
Ca, %
P, %
Mn, mg/kg
Fe, mg/kg
Cu, mg/kg
Zn, mg/kg
Se, g/kg
Mg, mg/kg
K, mg/kg
Na, mg/kg
I, mg/kg
Vitamin A, IU/kg
Vitamin D3, ICU/kg
Vitamin E, mg/kg

Amount
23.5
5.55
3100
132
2.66
5.84
0.85
0.50
99.9
76.7
13.9
78.9
112
1773
8406
1650
1.0
4849
900
54.8

Component
Vitamin K, mg/kg
Choline, mg/kg
Niacin, mg/kg
Pantothenic acid, mg/kg
Vitamin B6, mg/kg
Riboflavin, mg/kg
Thiamine, mg/kg
Folic acid, mg/kg
Vitamin B12, g/kg
Biotin, g/kg
Arginine, %
Glycine, %
Methionine, %
Cysteine, %
TSAA, %
Lysine, %
Tryptophan, %
Linoleic acid, %
Xanthophyll, mg/kg

Amount
0.7
1935
105.7
13.0
7.4
5.7
2.6
1.4
106
252
1.700
1.275
0.486
0.434
0.920
1.326
0.324
1.19
8.63

Table 4. Composition of cattle ration (cattle supplement for corn silage 2 LB/H/D)
Ingredient
Alfalfa meal, dehydrated, 17%
Soybean oil meal, solvent extracted
Urea, feed grade
Dicalcium phosphate
Salt
Calcium carbonate
a
Trace mineral premix (AN-02) (CA + SH)
b
Vitamin A and D3 premix (CA-1)
c
Vitamin E premix
Potassium chloride (DYNA K)
Total
a

b
c

Amount, %

Amount, lbs/ton

7.20
67.24
3.00
4.80
6.00
6.60
0.48
0.84
0.84
3.00

144.0
1344.8
60.0
96.0
120.0
132.0
9.6
16.8
16.8
60.0

100.0

2000.0

Trace mineral premix contains 2.50% Mn as manganese oxide, 0.07% I as potassium iodide, 0.30% Co as cobalt carbonate, 0.50% Cu as
copper oxide, and 20.00% Zn as zinc sulfate.
Each pound of vitamins A and D3 premix contains 2 000 000 USP units vitamin A and 225 750 USP units vitamin D3.
Each pound of vitamin E premix contains 20 000 IU vitamin E.

Table 5. Calculated analysis for cattle ration (cattle supplement for corn silage 2 LB/H/D)
Component
Dry matter, %
NE/M, mcal/kg
TDN, %
DIG protein, %
Ca, %
Cu, mg/kg
Fe, %
Mn, mg/kg
K, %
S, %
Vitamin A (total), IU/kg
Vitamin E (total), IU/kg
Vitamin D (total), IU/kg
Se, mg/kg

Amount
91.392
1.392
58.928
38.961
3.914
52.197
0.034
161.477
3.179
0.00
42026.400
370.440
4181.378
0.00

Component
ME, mcal/kg
NE/P, mcal/kg
Crude protein, %
Crude fiber, %
Co, %
I, mg/kg
Mg, %
P, %
Na, %
Zn, mg/kg
Vitamin A (added), IU/kg
Vitamin E (added), IU/kg
Vitamin D (added), IU/kg

Amount
2.131
0.917
44.441
6.384
0.097
3.372
0.224
1.435
2.667
961.238
37044.000
370.440
4181.343

Table 6. Composition of cattle heifer growing ration (CA95)

Ingredient
Ground corn, yellow
Soybean meal, 48% (solvent extracted, without hulls)
Molasses, cane
Distillers dried grain (corn)
Meat meal
a
BF 46S
Salt
Total
a

Amount, %

Amount, lbs/ton

63.00
10.00
10.0
9.00
4.60
2.80
0.60

1260.0
200.0
200.0
180.0
92.0
56.0
12.0

100.0

2000.0

BF 46S contains 23.8% Ca, 8.19% Cl, 1.4% Mg, 4.0% P, 7.25% K, 5.5% salt, 2.37% Na, 2.77% S, 9.2 mg/kg Co, 200 mg/kg Cu, 51 mg/kg I,
23 mg/kg Fe, 427 mg/kg Mn, 5.0 mg/kg Se, 667 mg/kg Zn, 550 KIU/kg vitamin A, 55 KIU/kg vitamin D3, 77 IU/kg vitamin E.

Table 7. Calculated analysis for cattle heifer growing ration (CA95)

Component
Dry matter, %
NE/M, mcal/kg
TDN, %
Crude fiber, %
Neutral DET fiber, %
Ca, %
Co, ppm
Fe, ppm
Mn, ppm
K, %
S, %
Se, ppm
Vitamin A (total), IU/kg
Vitamin E (total), IU/kg
Vitamin D (total), IU/kg

Amount
88.12
1.36
80.23
3.31
10.61
1.36
0.45
100.47
28.36
1.07
0.32
0.29
18200.00
19.13
1748.00

Component

Amount

ME, mcal/kg
NE/P, mcal/kg
Crude protein, %
Acid detergent fiber, %
Soluble protein, %
Cl, %
Cu, ppm
Mg, %
P, %
Na, %
Zn, ppm
I, ppm
Vitamin A (added), IU/kg
Vitamin E (added), IU/kg
Vitamin D (added), IU/kg

1.97
1.85
17.31
4.13
2.99
1.03
22.94
0.14
0.66
0.46
46.31
1.82
1745.00
2.45
1748.00

Table 8. Summary of resolution, retention time, and tailing for monensin LC collaborative study
Resolution factor (Rs)
Laboratory
A
B
C
D
E
F
G
H
I
J
Mean
RSD, %

Mon B Mon A Mon A Nar

Retention time, s
Monensin B

Monensin A

1.48
1.47
1.76
2.58
1.32
1.58
1.31
1.38
1.49
1.48

3.81
3.61
4.66
6.63
3.86
4.24
3.51
3.54
3.57
3.61

562
517

517
697
587
572
572
555
584

630
578
643
590
782
708
626
629
611
648

1.58
23.6

4.10
23.4

574
9.2

644
9.3

Tailing
Narasin
870
741

797
1018
1050
780
795
757
827
848
13.2

Monensin B

Monensin A

Narasin

1.02
1.38

1.28

1.48
1.02
1.25
1.09
1.04

1.08
1.31
1.20
1.25
1.22
1.23
1.22
1.23
1.15
1.13

1.19
1.17

1.20
1.31
1.39
1.19
1.29
1.17
1.18

1.20
14.9

1.20
5.5

1.23
6.4

Table 9. Summary of monensin results (g/ton) for control poultry and cattle rations and liquid feed supplement (LFS)
Laboratory
Sample

Monensin,
g/ton

Poultry

0.0

<0.4

0.0

<1

0.1

0.0

0.0

<10

0.0

<4

Cattle 1

0.0

<0.4

0.0

<1

0.4

0.1

0.0

<10

0.0

<4

Cattle 2

9.7

<0.4

0.1

<1

0.4

0.2

0.0

<10

0.0

<4

LFS

0.0

<0.3

0.2

<1

0.1

0.9

0.3

<10

0.2

<4

Table 10. Summary of premix results (mg/g) for monensin LC collaborative study
Laboratory
Sample
Coban premix
Rumensin premix
a

Monensin,
mg/g

Mean,
mg/g

RSD, %

132
176

134.0
191.0

125.1
182.0

108.0a
138.0a

134.0
197.0

192.7a
125.2a

130.1
198.4

130.5
189.2

125.0
181.0

130.0
189.0

133.0
195.0

130.2
190.3

2.8
3.4

Value was deleted from data set and not included in statistical evaluation on the basis of Cochran and Grubb procedures.

Table 11. Summary of monensin results (g/ton) for poultry rations, cattle rations and liquid feed supplements for
monensin LC collaborative study
Laboratory
Sample

Monensin,
g/ton

Poultry
Poultry
Poultry
Poultry
Cattle 1
Cattle 1
Cattle 2
Cattle 2
Cattle 2
Cattle 2
LFS
LFS
a
LFS
a
LFS

30
30
110
110
7
7
7
7
30
30
25
25
200
200

28.4
26.9
100.0
109.0
9.1
8.3
6.4
8.5
34.2
26.1
25.6
26.5
213.0
212.0

29.6
27.8
112.9
95.9
8.1
8.2
10.4
9.5
38.4
33.0
24.8
25.8
210.8
209.8

32.5
35.9
106.0
115.0
6.5
7.5
9.2
7.9
35.2
27.7
24.7
24.4
176.0
22639.0

33.2
34.7
117.0
121.0
8.3
8.9
8.9
9.5
41.3
28.7
33.0
32.2
173.0
262.0

39.9
30.0
123.7
118.5
8.7
8.3
11.0
7.8
36.2
27.5
29.2
28.8
241.6
248.5

31.3
34.9
105.1
122.7
12.1
9.4
11.0
8.3
36.7
27.9
27.2
27.9
40.5
218.4

36.5
33.2
122.3
116.6
6.9
8.9
9.0
8.6
32.7
31.2
28.3
28.2
226.9
225.5

27.0
36.6
106.0
101.0
9.1
7.9
10.4
9.3
32.7
26.8
23.8
11.9
55.4
133.0

29.0
34.0
133.0
140.0
9.0
8.0
8.0
10.0
32.0
30.0
26.0
27.0
216.0
222.0

31.0
35.0
115.0
116.0
9.8
8.8
7.0
8.8
36.0
34.0
26.0
4.6
210.0
59.0

Mean,
g/ton RSD, %
31.8
32.9
114.1
115.6
8.8
8.4
9.1
8.8
35.5
29.3
26.9
23.7
176.3
2442.9

12.3
10.4
9.0
10.5
17.6
6.6
17.7
8.4
8.1
9.1
10.1
36.2
40.2
290.5

Values for 200 g/ton were deleted from data set and not included in statistical evaluation on the basis of Cochran and Grubb procedures.

Figure 997.04A. Chromatogram of typical system suitablility sample (monensin and narasin).

Figure 997.04B. Chromatogram of Rumensin 80 type A medicated article.

Figure 997.04C. Chromatogram of monensin type C medicated feed.

Figure 1. Diagram of LC postcolumn derivatization

system: S1 = LC mobile phase; P1 = pulse-dampened
LC pump operating at 0.7 mL/min; A = autosampler
equipped with 200 L injection loop; C = LC column; T =
90 tee plumbed such that inlet flows directly oppose
each other; P2 = reagent pump operating at 0.7 mL/min;
S2 = vanilin reagent; CO = 0.020 in. 20 ft. stainless
steel coil enclosed in a 98.0C heater; D = variablewavelength absorbance detector (Kratos Model 757 or
equivalent) operating at 520 nm with rise time of 1.0 and
range of 0.2 (or as needed); and R = recorder or
integrator (manual or computer).