Analysis of gentian violet residue in tissue

A process for the analysis of gentian violet in animal or poultry tissue, animal or poultry manure, animal or poultry feed, or eggs. The gentian violet is quantitatively determined.

BACKGROUND OF THIS INVENTION 
1. Field of This Invention 
This invention further relates to processes for the analysis of residual 
gentian violet in tissue, eggs, manure or feed. 
2. Prior Art 
U.S. Pat. No. 3,915,637 involves a process for the analysis of the residual 
gentian violet in feed, tissue, eggs or manure. The process includes 
extracting substantially all of the fat in the feed, tissue, eggs or 
manure using a fat solvent. (The extraction is preferably done at room 
temperature. The fat solvent is separated from the feed, tissue, eggs or 
manure, preferably, by means of centrifuging.) The fat-extracted feed or 
manure is dried. A known amount of acidified ethanol is admixed with the 
fat-extracted feed, eggs, tissue or manure, and the resultant admixture is 
left standing (preferably for 48 hours). The acidified alcohol is 
separated from the resultant admixture. The amount of gentian violet in 
the acidified alcohol is determined by spectrophotometrical comparison 
with acidified alcohol standards containing set amounts of gentian violet. 
The extraction, separation and determination steps are repeated until no 
gentian violet is found to be present in the acidified alcohol, the total 
of the gentian violet found from the determination steps being the total 
amount of gentian violet present in the feed, eggs, tissue or manure. 
Preferably the fat extraction step involves first extracting the fat from 
the manure, tissue, eggs or feed at least once with hexane. The preferred 
fat solvent is petroleum ether, when a one-step extraction process is 
used, and is the use of petroleum ether, in the first step and the use of 
hexane in the second step, when a two-step extraction step is used. Other 
fat solvents can be used. Preferably the acidified alcohol contains 1 ml. 
of concentrated HCl per 100 ml. of ethanol. Other acids such as 
concentrated sulfuric acid and concentrated nitric acid can be used in 
place of the concentrated HCl. Other suitable solvents can be used in 
place of the acidified alcohol. The determinations can be made by any 
conventional method, but preferably are made using a spectrophotometer, 
such as, a Turner spectrophotometer. 
The patent also involves a process for checking the accuracy of a process 
for the analysis of residual gentian violet in feed or manure of animal or 
poultry. 
BROAD DESCRIPTION OF THIS INVENTION 
An object of this invention is to provide a more sensitive and simplified 
analytic process for quantitatively determining the gentian violet content 
of materials, such as, animal or poultry tissue. A further object of this 
invention is to provide a process for checking the accuracy of such 
analytic process. Other objects and advantages of this process are set out 
herein or obvious herefrom to one ordinarily skilled in the art. 
The process of this invention achieves the objects and advantages of this 
invention. 
This invention involves a process for the analysis of gentian violet in a 
material. The process includes finely particulating a known amount of 
material containing gentian violet, unless such material is already in a 
finely divided state. A known amount of acidified ethanol is admixed with 
the finely particulated tissue and the resultant admixture is allowed to 
stand. The acidified ethanol removes the gentian violet from the material. 
The acidified ethanol is separated from the admixture by centrifugation. 
Any spectrophotometrical interfering substance is removed from the 
acidified ethanol by passing the acidified ethanol through a chromatograph 
column. The amount of gentian violet in the acidified ethanol and the 
background measurement of the acidified ethanol is determined by 
spectrophotometrical means. The gentian violet in said acidified ethanol 
is quenched by adding to the acidified ethanol a chemical substance which 
selectively renders the gentian violet non-detectable by 
spectrophotometric means without disturbing the spectrophotometric 
background of the acidified ethanol. The background measurement of the 
acidified ethanol is determined by spectrophotometrical means. The 
quantitative amount of gentian violet in the material is calculated using 
the two spectrophotometrical measurements. 
Preferably the material containing gentian violet is animal tissue, poultry 
tissue, eggs, animal feed, poultry feed, animal manure or poultry manure. 
Preferably the acidified ethanol was acidified by HCl. Preferably the two 
spectrophotometric measurements are made at 590 m.mu.. 
This invention also includes a process for checking the accuracy of the 
process for the analysis of gentian violet in a material. The checking 
process involves finely particulating a known amount of a material known 
not to contain any gentian violet, unless such material is already in a 
finely divided state. A known amount of at least one aqueous solution 
containing a known amount of gentian violet is added to a known amount of 
the material known not to contain any gentian violet to form at least one 
spiked sample. A known amount of acidified ethanol is admixed with the 
finely particulated tissue and the resultant admixture is let stand. The 
acidified ethanol removes the gentian violet from the material. The 
acidified ethanol is separated from the admixture by centrifugation. Any 
spectrophotometrical interfering substance is removed from the acidified 
ethanol by passing the acidified ethanol through a chromatograph column. 
The amount of gentian violet in the acidified ethanol and the background 
measurement of the acidified ethanol is determined by spectrophotometrical 
means. The gentian violet in the acidified ethanol is quenched by adding 
to the acidified ethanol a chemical substance which selectively renders 
the gentian violet non-detectable by spectrophotometric means without 
disturbing the spectrophotometric background of the acidified ethanol. The 
background measurement of the acidified ethanol is determined by 
spectrophotometrical means. The quantitative amount of gentian violet in 
the spiked material is calculated using the two spectrophotometrical 
measurements. The amount of gentian violet in the spiked sample gives the 
accuracy of the procedure for the analysis of gentian violet in the spiked 
material by direct comparison with the known amount of gentian violet 
placed in the spiked sample. 
Preferably at least two spiked samples are prepared, each of the spiked 
samples having a different amount of gentian violet. Preferably the 
material containing gentian violet is animal tissue, poultry tissue, eggs, 
animal feed, poultry feed, animal manure or poultry manure. 
An advantage of this invention over the prior art is that the process of 
this invention is much more accurate and sensitive than the prior method 
or methods. The best prior art method is that of U.S. Pat. No. 3,915,637. 
This invention achieves far more accuracy and sensitivity than the process 
of U.S. Pat. No. 3,915,637 by the use of at least two improvements. The 
first improvement involves the passage of the initial extraction solution 
through a chromatographic column to trap all of the interfering materials 
to provide a clear eluant for spectrophotometric analysis. The second 
improvement involves quenching the gentian violet in the eluant to allow 
for quantitative calculation of the gentian violet recovery. This 
quenching technique eliminates the question of background interference as 
only the gentian violet is quenched from the eluant. 
The sensitivity of the process of this invention, due mostly to the two 
above-described process improvements, is greatly improved over that of the 
process of U.S. Pat. No. 3,915,637 With a Turner spectrophotometer, or its 
equivalent, utilizing 13 mm cuvettes, the analytical process of U.S. Pat. 
No. 3,915,637 has a sensitivity at most of 0.1 ppm gentian violet, whereas 
the analytical process of this invention has a sensitivity of 0.1 to 0.04 
ppm gentian violet. With a Beckman D.U. spectrophotometer, or its 
equivalent, utilizing 100 mm cells, the analytical process of U.S. Pat. 
No. 3,915,637 has a sensitivity at most of 0.01 ppm gentian violet, 
whereas the analytical process of this invention has a sensitivity of 0.01 
to 0.005 ppm gentian violet. So the analytical process of this invention 
is eminently applicable to the quantitative determination of the gentian 
violet content of or residue in tissue samples containing 0.1 ppm or less 
gentian violet (depending on the spectrophotometer and cells utilized in 
the determination). This is also so with eggs and manure. 
The process of this invention achieves an increase of sensitivity to a 5 
ppb level. 
The process of this invention does not require an involved fat extraction 
step (using fat solvents, e.g., petroleum ether and hexane in sequence, 
followed by a separation of the fat solvent). This invention uses the much 
more accurate chromatograph column to remove the spectrophotometric 
interfering substances after the acid-ethanol extraction step. 
An advantage of this invention is that no process is necessary for checking 
the accuracy of the analytical process of this invention. Such a checking 
can be used if desired, but is not necessary due to the inherent accuracy 
of the invention process. 
DETAILED DESCRIPTION OF THIS INVENTION 
This invention can even be used to detect gentian violet residues in 
tissue. In such the gentian violet has reacted with the tissue. Spiked 
tissues may not achieve this chemical bonding. 
As used herein, the term "poultry" means domestic fowl including chickens, 
ducks, turkeys, geese, etc. As used herein, the term "animal" includes 
domestic pigs, other swine, cattle, sheep, goats, rabbits, etc. The term 
animal includes humans and non-human animals. 
As used herein, the term "gentian violet" is hexamethylpararosaniline 
chloride or a mixture of at least 90 percent of hexamethylpararosaniline 
chloride with minor portions of pentamethylpararosaniline chloride and/or 
tetramethylpararosaniline chloride, and preferably meeting all 
specifications of quality for U.S.P. grade as listed in the United States 
Pharmacopiea XIV. This assures that none of the deadly heavy metals is 
left as a residue in animal or poultry tissue that is to be consumed by 
man. 
Gentian violet is a dark green powder or greenish, glistening pieces with a 
metallic luster. Gentian violet is soluble in water, chloroform and 
ethanol. Other names for gentian violet are methyl rosaniline chloride and 
crystal violet. 
Gentian violet, both as a contaminant in water and as a residue in poultry 
manure, is biodegradable in the presence of the ultra-violet rays of 
sunlight. 
Another name for Candida albicans is Monilia albicans. Candida albicans, a 
yeast-like organism, is the usual cause of moniliasis pneumonia, 
meningitis and other forms of moniliasis. It is normally saprophytic but 
may become pathogenic after the administration of certain antibiotics. 
(Antibiotics are often used in relatively large amounts in animal and 
poultry feeds. The use of the antibiotics allows molds and fungi to grow.) 
The use of gentian violet in water and feed is useful in eliminating and 
preventing internal infestation of Candida albicans, for example, in the 
intestines, and infestation of Candida albicans in drinking water. 
Gentian violet inhibits the growth of and destroys Candida albicans without 
materially reducing the growth of and activity of the normal coliform 
bacteria present in the intestinal tract, which is necessary in 
synthesizing important nutritional factors. without 
U.S. Pat. Nos. 3,916,027 and 3,915,637 disclose the use of gentian violet 
as an additive to animal and poultry feed for the prevention and treatment 
of candidiasis caused by Candida albicans. As a result there is a buildup 
of gentian violet residue in the tissue of any animals and poultry 
consuming feed containing gentian violet. The residue levels involved in 
consumption at the levels given in U.S. Pat. No. 3,916,027 are not toxic 
or harmful to the consuming animal or poultry. The consuming animals or 
poultry are put on a withdrawal feed period (i.e., feed which does not 
contain any gentian violet) to lower the gentian violet residue level when 
the animals are to be consumed by man. It is important to get as low a 
gentian violet level as possible in the animal or poultry tissue--the 
continued consumption of a low (even very low) amount of a chemical may 
sensitize a human to the chemical. This means the subsequent use of 
gentian violet as a medicine or medicament for humans who had been so 
sensitized could be quite harmful to such humans from severe adverse 
reactions. In view of this it is important to have an analytic procedure 
to detect much less than 0.1 ppm gentian violet residue (maximum level 
allowed by the F.D.A.) in animal or poultry tissue that is to be consumed 
by man. Lower levels are required by the F.D.A. in eggs, so the accuracy 
of this procedure is also needed in the egg field. The manure field is 
necessary, among other reasons, to get a complete accounting of the 
gentian violet consumed by animals or poultry. 
The gentian violet is preferably used in the form of a premix concentrate, 
which can be added to the complete feed as needed. The most preferred 
premix concentrate contains corn cob fractions, white mineral oil, 
diatomaceous silica (e.g., Micro-Cel E), distilled water and 1.55 percent 
of gentian volet. 
The concentration of gentian violet in the premix concentrate can easily be 
as high as 60 percent; it is preferably between 0.1 and 10 percent and is 
most preferably 1.55 percent. A useful gentian violet premix concentrate 
is premix ViGen (concentrate) obtainable from A.H.P., Inc., Gainsville, 
Ga. 
The premix concentrate can contain any material that is used in a complete 
poultry feed or animal feed. The premix concentrate can be used in the 
form of a liquid or solid admixture. The liquid is best in the form of a 
very viscous suspension or similar semi-fluid. Preferably a solid premix 
concentrate is used even though it may contain a liquid material such as 
white mineral oil which reduces the dust factor in the premix. 
The premix concentrate is used for treatment of Candida albicans by adding 
it to the complete poultry or animal feed for a period of about 7 days, 
although the time can be more or less as needed. When the premix 
concentrate contains 1.55 percent of gentian violet, preferably 2 pounds 
of the premix is ued per ton of complete poultry or animal feed. That 
reflects a preferred concentration of about 0.00155 percent of gentian 
violet in the complete poultry feed. Effective concentrations of gentian 
violet are quite wide, but an example of the range is 0.0001 to 0.01 
percent of gentian violet per ton of complete poultry or animal feed. 
The above treatment times and concentrations in the complete poultry or 
animal feed apply even when the gentian violet is directly admixed with 
the complete poultry or animal feed without going through the intermediate 
premix concentrate form. 
The premix is used for prevention of Candida albicans by adding it to the 
complete poultry or animal feed on a continuous basis. (After treatment 
with the premix, it should be used on a continuous basis to achieve 
prevention.) When the premix contains 1.55 percent of gentian violet, 
preferably one pound of premix is used per ton of complete poultry or 
animal feed. That reflects a preferred concentration of about 0.00077 
percent of gentian violet in the complete poultry or animal feed. 
Effective concentrations of gentian violet are quite wide, but an example 
of the range is 0.00005 to 0.005 percent of gentian violet per ton of 
complete poultry or animal feed. 
The above treatment times and concentrations in the complete poultry or 
animal feed apply even when the gentian violet is directly admixed with 
the complete poultry or animal feed without going through the intermediate 
premix concentrate form. 
Any complete poultry feed or basal poultry feed can be used. It can 
contain, for example, ground yellow corn, soybean oil meal, steamed bone 
meal, ground limestone, iodized salt, manganese sulfate, Vitamin A oil, 
dry Vitamin D-3, riboflavin, Vitamin B-12 and niacin. It can also contain, 
for example, fish meal and meat meal. 
Any complete amimal feed or basal animal feed can be used; it can contain, 
for example, any of the following ingredients: mogul starch, iodized salt, 
dry Vitamin D-3, riboflavin, Vitamin B-12, niacin, meat meal, calcium 
pantothenate, cracked or milled grains such as corn, wheat, oats, barley 
and the like, dried molasses, dried sorghum, soybean meal, cottonseed 
meal, peanut meal, fish meal, essential amino acids such as lysine, 
peptides and polypeptides containing essential amino acids, casein, soya 
bean protein, vitamins such as Vitamins A, E and K, mineral nutrients such 
as sodium chloride, ferrous salts, magnesiun sulfate and calcium salts, 
proteins, buffers, dextrose, sucrose, lactose, maltose, corn syrup solids, 
hydrolyzed cereal solids, hay, etc. 
An exemplary cattle feed is 73 percent rolled shelled corn, 20 percent 
ground corn cobs, and a supplement containing soybean meal, alfalfa meal, 
cane molasses, urea, salt, dicalcium phosphate, Vitamin A concentrate and 
Vitamin D concentrate. 
The gentian violet may be consumed as a poultry water sanitizer. It aids in 
the reduction of Candida albicans contaimination in water lines and 
poultry watering equipment. It may be put directly into polutry water to 
treat and prevent candidiasis. 
The gentian violet is preferably used in the form of a liquid premix 
concentrate, which can be added to the poultry water on a regular basis, 
for example, once a week. It can also be made up into a stock solution and 
added to the poultry water. The most preferred concentrate contains water 
and 0.3875 percent of gentian violet, but the concentration usually ranges 
from 0.05 to 5 percent, although any concentration can be used. The key is 
to get a sufficiently high concentration in the poultry water itself. The 
concentration of gentian violet in the poultry water itself should be 
between 0.001 and 0.05 percent. 
The treatment period is preferably one day every week, but can be on any 
basis as needed to achieve the desired effect. 
The gentian violet can be added directly to the poultry water, in which 
case the above treatment times and concentrations also apply. 
The pertinent portions of applicant's copending application Ser. No. 
625,693, filed Oct. 24, 1975, entitled "Feed Composition and Process of 
Sparing Poultry from the Effects of Aflatoxin", is incorporated herein by 
reference. 
U.S. Ser. No. 625,693 discloses a method for sparing an animal or poultry 
from the toxic effect of a mycotoxin in animal or poultry feed. The method 
includes feeding a complete feed to the animal or poultry, the complete 
feed being comprised of (i) gentian violet, and (ii) the remainder inert 
ingredients. The gentian violet spares the animal or poultry from the 
effect of mycotoxin in the feed as a contaminant. The gentian violet is 
present in the feed in an amount between 0.00077 and 0.01 percent by 
weight, based on the weight of the complete feed. 
A premix concentrate can be added to the feed. The premix contains between 
0.1 and 10 percent by weight of gentian violet and the remainder inert 
ingredients. Enough of the premix concentrate to the other ingredients of 
the feed is used to obtain the recited amounts of gentian violet in the 
feed. 
Preferably the feed contains 0.00155 to 0.00232 percent by weight of 
gentian violet, based on the weight of the complete poultry feed. 
Such is quite useful for feeds that are contaminated with as much as 10 
p.p.m. or more of mycotoxin. 
The sparing effect of gentian violet appears to be a linear function of the 
concentration of the aflatoxin in the feed. 
The growth of the animal or poultry taking mycotoxin-contaminated poultry 
or animal feed containing gentian violet at the claimed levels is 
increased at a rate significantly faster than if the animal or poultry had 
not been fed such mycotoxin-contaminated feed containing gentian violet. 
Gentian violet works as a growth promoter when the animal or poultry are 
even consuming feed which is contaminated with aflatoxin. 
The levels of gentian violet used in this invention are low enough not to 
damage the consuming animal and not to produce an unacceptable residue in 
food from the animal. The level of residue is extremely low. 
Gentian violet is essentially non-absorbable and stable in the pH range of 
the digestive tract of animals and poultry. 
Any complete poultry feed or basal poultry or complete animal feed or basal 
animal feed can be used. 
The greatest mycotoxin effect sparing is achieved in young, maturing 
animals or poultry, although excellent mycotoxin effect sparing is 
achieved in grown mature animals or poultry. 
An important feature is that there is little or no residue of the gentian 
violet in the tissue of the animals fed the compositions this is 
apparently so due, in part, to the levels of gentian violet used in the 
compositions of this invention. This means that there is no toxicological 
danger to animals or humans from ingestion of the edible tissues of 
animals which have been fed the compositions. 
Applicant's claimed concentration ranges in the animal and poultry feeds 
are important re efficacy, mycotoxin effect sparing in poultry or animals. 
If amounts lower than applicant's claimed ranges are used, there is little 
or no efficacy in aflatoxin effect sparing. If an amount of gentian violet 
is used which is higher than applicant's claimed range (in feed), toxic 
effects in the poultry can be encountered. The toxic effect can kill the 
poultry or cause a decrease in the weight gain of the poultry, which 
represents a serious economic loss to commercial poultry raisers (and 
loses the advantage of the gentian violet being a growth promoter). The 
residue build up is a potential health hazard when such higher levels are 
used. 
The pertinent portions of applicant's copending application Ser. No. 
625,873, filed Oct. 24, 1975, entitled "Growth Promoter and Process", is 
incorporated herein by reference. The pertinent portions of applicant's 
copending U.S. Ser. No. 595,876, filed on July 14, 1975, entitled "Growth 
Promoter and Process", is incorporated herein by reference. 
For the best effects the feed composition of this invention is fed to 
poultry, hogs or cattle. 
Preferably the mix of preparing the feed involves preparing a premix 
concentrate for addition to the feed which consists only of between 0.1 
and 10 percent by weight of gentian violet and the remainder inert 
ingredients, and then adding enough of the premix concentrate to the other 
ingredients of feed to obtain the recited amount of gentian violet in the 
feed. Preferably inert ingredients used in the premix concentrate are 
diatomaceous silica, corn cob fractions and vegetable oil. Most preferably 
the feed contains 0.00155 percent by weight of gentian violet, based on 
the weight of the complete feed. 
The gentian violet is preferably used in the form of a premix concentrate, 
which can be added to the complete feed as needed. The most preferred 
premix concentrate contains corn cob fractions, white mineral oil, 
diatomaceous silica (e.g., Micro-Cel E), distilled water and 1.55 percent 
of gentian violet. 
The concentration of gentian violet in the premix concentrate can easily be 
as high as 60 percent; it is preferably between 0.1 and 10 percent and is 
most preferably 1.55 percent. A useful gentian violet premix concentrate 
is premix ViGen (concentrate) obtainable from A.H.P. Inc., Gainesville, 
Ga. 
The premix concentrate can contain any material that is used in a complete 
or basal animal or poultry feed. The premix concentrate can be used in the 
form of a liquid or solid admixture. The liquid is best in the form of a 
very viscous suspension or similar semi-fluid. Preferably a solid premix 
concentrate is used even though it may contain a liquid material such as 
white mineral oil which reduces the dust factor in the premix. 
The premix concentrate is used by adding it to the complete poultry feed as 
long as growth stimulation is wanted. When the premix concentrate contains 
1.55 percent of gentian violet, preferably two pounds of the premix is 
used per ton of complete poultry feed for growth stimulation of animals or 
poultry-- this reflects a preferred concentration of about 0.00155 percent 
of gentian violet in the complete poultry feed. Effective concentrations 
of gentian violet are within the range of 0.00077 to 0.005 percent of 
gentian violet per ton of complete poultry feed. The preferred concentrate 
range of gentian violet is from 0.00085 to 0.01 percent of gentian violet 
per ton of complete poultry feed. Most preferably female poultry are fed 
at a gentian violet level of 0.00155 percent per ton of complete poultry 
feed; and most preferably male poultry are fed at a gentian violet level 
of 0.00385 percent per ton of complete poultry feed. 
The above concentrations in the complete poultry feed apply even when the 
gentian violet is directly admixed with the complete poultry feed without 
going through th intermediate premix concentrate form. 
The premix is used for growth stimulation by adding it to the complete feed 
on a continuous basis. 
Any complete poultry feed or basal poultry feed or complete animal feed or 
basal animal feed can be used. 
The greatest growth stimulation is achieved in young, maturing animals or 
poultry, although excellent growth stimulation is achieved in grown mature 
animals or poultry. 
An important feature is that there is little or no residue of the gentian 
violet in the tissue of the animals fed the compositions-- this is 
apparently so due, in part, to the levels of gentian violet used in the 
composition. This means that there is no toxicological danger to animals 
or humans from ingestion of the edible tissues of animals which have been 
fed the compositions. 
Applicant's claimed concentration ranges in the animal and poultry feeds 
are important re efficacy, growth stimulation and toxicity in poultry or 
animals. If amounts lower than applicant's claimed ranges are used, there 
is little or no growth stimulation of animals or poultry. If an amount of 
gentian violet is used which is higher than applicant's claimed range (in 
feed), then toxic effects in the poultry can be encountered. The toxic 
effect can kill the poultry or cause a decrease in the weight gain of the 
poultry, which represents a serious economic loss to commercial poultry 
raisers (and loses the advantage of the gentian violet being a growth 
promoter). The residue build up is a potential health hazard when such 
higher levels are used. 
U.S. Ser. No. 625,873 discloses the use of an effective amount of at least 
one specific-acting drug along with the gentian violet in the animal or 
poultry feed. 
The eggs, feed, tissue or manure (A known amount) is finely particulated, 
if necessary. In the case of tissue or eggs, the process preferably 
includes finely particulating a known amount of egg or tissue. The tissue 
can be, for example, muscle, skin, fat, kidney and liver. The tissue or 
egg is preferably finely particulated by gine grinding. 
The gentian violet can be residual or added (spiked). 
No fat extraction step (using a fat solvent) is necessary at all or before 
the acid-alcohol extraction step. 
This process proceeds with an acid-alcohol extraction of the gentian violet 
from the tissue, egg, manure or feed. A known amount of acidified ethanol 
is admixed with the tissue, eggs, manure or feed, then the resultant 
admixture is let stand (preferably at room temperature in the dark for 
about 12 to 24 hours). Preferably the acidified alcohol contains 1 ml of 
the concentrated HCl per 100 ml of ethanol. Other acids, such as 
concentrated sulfuric acid and concentrated nitric acid, can be used in 
place of the concentrated HCl. The acidified alcohol is separated from the 
resultant admixture (preferably by centrifuging). (It is not necessary to 
repeat the acidified alcohol extraction step.) 
Other suitable solvents can be used in place of the acidified ethanol. 
Straight ethanol is not a suitable alternative solvent for eggs and tissue 
since it only extracts at most about 50 percent of the gentian violet 
residue in eggs or tissue-- acidified alcohol removes 99 percent or more 
of the gentian violet residue in eggs or tissue. 
The acid ethanol extraction solution is passed through a chromatographic 
column to trap all of the interfering materials to provide a clear eluant 
for subsequent spectrophotometric analysis. The fatty substances are 
removed-- the need to use a fat solvent is eliminated. 
The gentian violet quenching step is achieved with a chemical substance 
which selectively renders the gentian violet non-detectable by 
spectrophotometric means without disturbing the spectrophotometric 
background of the acidified ethanol solvent solution. such chemical 
substance only quenches the gentian violet. 
The quenching agent has to be a strong oxidizing agent. The most preferred 
quenching agent is sodium hypochlorite (NAOCl). Other hypochlorites, such 
as calcium hypochlorite, potassium hypochlorite, barium hypochlorite, can 
be used as the quenching agent-- alkali metal and alkaline earth metal 
hypochlorites are particularly useful. The hypochlorites are useful 
because no salt is formed and hence no precipitation thereof. For example, 
the HCl in the acid-alcohol solvent reacts with a quenching agent such as 
NaOH to form NaCl. which precipitates leaving a cloudy solution. But NaCl 
does not react with HCl to form a precipitating salt-- it reacts with the 
gentian violet to render the gentian violet colorless. 
The amount of quenching agent used is important. If too much quenching 
agent is used, the tissue will be oxidized and this will change the 
background. If too little quenching agent is used, not all of the gentian 
violet will be oxidized. So an effective amount of quenching agent, with 
such limits, is used. 
If it is desired to measure the amount of gentian violet is a tissue sample 
in which the amount of gentian violet is completely unknown, a background 
reading is made. Then the quenching agent is added in increments until two 
identical readings are obtained. At this point it is known that all of the 
gentian violet has been quenched. 
The procedure of this invention allows for the leaching of the protein 
bound gentian violet form the tissue samples with minimum background 
interference from other color components contained in the tissue samples. 
When the tissues are subjected to digestion in the appropriate solvent, 
the gentian violet is quantitatively released from the protein. 
Gentian violet exhibits a characteristic absorption at 590 m.mu. which is 
suitable for the quantitative measurement of the compound. 
To repeat, in this invention the gentian violet content of poultry tissues 
(for example) is determined spectrophotometrically. This procedure 
consists of acid alcohol digestion of the tissue samples to leach the 
gentian violet bound to the protein fraction of the tissues, passage of 
the solution through a chromatographic column to clarify it, and 
spectrophotometric measurement of the gentian violet content of the final 
solution. This treatment of the tissue samples serves to isolate the 
gentian violet from material present in the tissues which might interfere 
in the spectrophotometric measurement. 
Preferably the steps of this process are conducted at room temperataure and 
pressure, but higher and lower temperatures can be used. 
The analysis method of this invention can also be used to quantitatively 
determine the presence of any substituted benzophenone azoanilide dyes or 
rosaniline base dyes other than the gentian violet in tissue, eggs, feed 
or manure, provided such other dye can be removed from the tissue, eggs, 
feed or manure by the acid - ethanol solvent. Such other dyes are also 
known as triaminotritane dyes related to rosaniline. The 
--N(CH.sub.3).sub.2 groups can be replaced with other auxochrome groups 
such as --OH, --NH.sub.2, --N(C.sub.2 H.sub.5).sub.2, etc. The other 
various substituents, and the substituents on gentian violet, can be 
located at any position on the three benzyl rings. The other dyes must 
have at least one amino chloride group and/or substituted amino chloride 
group. 
Examples of such other substituted benzophenone azoanilide dyes are: 
pentamethylpararosaniline chloride; tetramethylparosaniline chloride; 
methyl green or light green, which is the methyl chloride addition product 
of gentian violet; ethyl green, which is the ethyl chloride addition 
product of gentian violet; sulfonic acids of the rosanilines, such as, 
water blue and patent blue; phenylated rosanilines, such as, triphenyl 
fuchsin, aniline blue, diphenylamine blue and spirit blue; pararosaniline; 
mixtures of pararosaniline with its methyl homologs, such as fushsins and 
magentas. More broadly, any triphenylmethane dye, or for that matter any 
dye that contains an amino group or substituted amino group can be 
quantitatively determined as long as it can be removed from the tissue, 
feed, eggs or manure by the acid - ethanol solvent. Examples of such 
triphenylmethane dyes are the malachite green series of dyes, such as, 
malachite green. 
Most of such aniline dyes are rather toxic, but can be readily used in 
feeds, etc. if they are buffered by any generally non-toxic conventional 
buffering system to reduce their toxicity. 
All parts, percentages and ratios are on a weight basis, unless otherwise 
stated or otherwise obvious herefrom to one ordinarily skilled in the art.

EXAMPLE 1 
This example involves the spectrophotometrical determination of the gentian 
violet content (as a residue) of poultry tissue. 
The following reagents were used: 
a. alcohol, ethyl -- 95 percent denatured EX 280, L 376, Matheson, Coleman 
and Bell. 
b. acid, hydrochloric, concentrated -- R. G., J. T. Baker. 
c. Alumin AR 0 100 to 200 mesh, activity Grade-4, chromatographic Sorbent, 
Mallinckrodt Chemical Works. 
d. water, distilled. 
e. 0.26 percent aqueous sodium hypochlorite solution. 
The apparatus included: a Waring blender, variable speed; an analytical 
balance -- Ainsworth chainomatic type LCB; an analytical balance -- 
Mettler type P162; glass beakers -- 50 to 100 ml capacity; a spectrum 
spatula -- 8 inches; volumetric pipettes --0.01 ml, 0.1 ml, 0.5 ml, 1.0 ml 
and 5.0 ml; a magnetic mixer; magnetic stirring bars -- 3/4 inch; 
centrifuge tubes -- 60 ml glass; centrifuge -- speed to 3,000 rpm -- 
capable of accommodating 60 ml tubes; graduated cylinders -- 50 ml; 
Erlenmeyer flasks, 125 ml with ground glass stoppers; Turner 
spectrophotometer; 13 mm matched cuvetts; spectrophotometer -- Beckman 
D.U. quartz -- with attachment for 100 mm cells; spectrophotometer cells 
-- 100 mm -- Beckman 580021; and chromatographic columns -- 10 mm Fabco 
No. 4280 with 100 ml reservoir (or equivalent) equipped with stopcock. 
All phases of the analytic procedure of this invention must be conducted 
under conditions of diffused light. Gentian violet is extremely sensitive 
to decomposition by the ultra violet rays of sunlight. In this example, 
the laboratory was illuminated entirely by artificial light, with the 
source of light at least 2 meters away from the work areas. 
The tissue samples were protected from excess heat, as heat crystallizes 
the tissues and imparts excess background color to the extraction 
solutions. All procedures of this example were carried out at room 
temperature (68.degree. to 72.degree.F.). 
An acid alcohol solution was prepared by adding 20 ml of concentrated 
hydrochloric acid to 2,000 ml of 95 percent ethyl alcohol (denatured). 
A gentian violet stock solution was prepared as follows: Using the 
Ainsworth chainomatic analytical balance, 0.104 gm of crystalline gentian 
violet (USP) was accurately weighed out and quantitatively transferred to 
a 1000 ml volumetric flask and q.s. to 1000 ml with distilled water. A 
magnetic stirring bar was inserted into the flask and the gentian violet 
solution was mixed on a magnetic mixer for 1 hour. This solution contained 
100 .mu. g of gentian violet per ml (100 ppm). 
Gentian violet-acid alcohol standard solutions were prepared as follows: 
Five standard dilutions of gentian violet in acid alcohol were prepared 
and the absorbence (O.D.) for each dilution was determined. The absorbence 
of the 5 dilutions were plotted on a graph to determine the linearity of 
the gentian violet curve. 
2 ml of 100 ppm aqueous g.v. solution q.s. to 100 ml with ac-EtOH = 2 ppm 
1.5 ml of 100 ppm aqueous g.v. solution q.s. to 100 ml with ac-EtOH = 1.5 
ppm 
1.0 ml of 100 ppm aqueous g.v. solution q.s. to 100 ml with ac-EtOH = 1.0 
ppm 
0.5 ml of 100 ppm aqueous g.v. solution q.s. to 100 ml with ac-EtOH = 0.5 
ppm 
0.1 ml of 100 ppm aqueous g.v. solution q.s. to 100 ml with ac-EtOH = 0.1 
ppm 
The spectrophotometer was set at 590 m.mu. and zeroed using an acid alcohol 
blank. The absorbence of the 5 standard dilutions in the 13 mm cuvette was 
determined. The O.D. of the 1 ppm dilution in the 13 mm cuvette was 
recorded for use in calculation of the gentian violet recovery. (If method 
sensitivity below 0.1 ppm is desired, the O.D. of the 1 ppm standard in 
the 100 mm cell is recorded and the Beckman D.U. is used to make the 
determinations.) 
A sodium hypochlorite quenching solution was prepared as follows: 0.26 gm. 
of sodium hypochlorite was weighed into a 250 ml Erlenmeyer flask and q.s. 
to 100 ml with distilled water. This solution was 0.26 percent NaOCL. If 
sodium hypochlorite is not available in the laboratory, this solution can 
be made from any standard household bleaching solution containing sodium 
hypochlorite (i.e., Clorox) at the 5.25 percent concentration. 1 ml 5.25 
percent NaOCl q.s. to 20 ml with distilled water to give 20 ml of 0.26 
percent NaOCl. 
The tissue samples were prepared by grinding the tissues in a Waring-type 
blender to reduce the tissues to the smallest particle size possible. 
Using a Mettler P162-type balance, 5 gm samples of the finely ground 
tissues were weighed (in triplicate) into Erlenmeyer flasks. 
Gentian violet was extracted from tissue samples as follows: 50 ml of 
acid-alcohol were added (using a graduated glass cylinder) to each of the 
samples in the flasks. A 3/4 inch magnetic stirring bar was inserted into 
each sample. The flasks were stoppered. The samples were placed on the 
magnetic mixers and mixed for 1 hour. After mixing, the samples were set 
in the dark to digest for 12 hours (overnight). After 12 hours of 
digestion the samples were again placed on the magnetic mixer and mixed 
for 1 hour. The contents of the sample flasks were poured into 60 ml glass 
centrifuge tubes and centrifuged at 2400 rpm for 10 minutes. 
Chromatographic column clearing of the centrifuged samples was achieved as 
follows: Each column was equipped with a glass stop-cock, if the column 
was not so equipped. A small glass-wool pledget was inserted firmly in the 
bottom of the column to fill the bottom curve of the column. 5 to 6 grams 
of the Alumin AR sorbent was added to each column. The supernatant liquid 
was carefully decanted from the centrifuge tube into the chromatographic 
column. A 50 ml beaker was placed under each column. The stop-cock was 
opened and the first eluant from the column was collected until a minimum 
of 20 ml was collected. (This first eluant ordinarily was yellow in color 
but contained the extracted gentian violet.) 
Spectrophotometric determination of the gentian violet concentration from 
the tissue samples was determined as follows: 5 ml of each of the eluants 
was pipetted into the 13 mm cuvettes. The spectrophotometer was set at 590 
m.mu.. The spectrophotometer was zeroed using an acid alcohol blank in the 
13 mm cuvette. It was established that all cuvettes were matched. The 
absorbence of each of the samples was read and the optical density (O.D.) 
reading was recorded. After the initial absorbence was determined and 
recorded, 0.01 ml of the 0.26 percent NaOCl solution was pipetted into 
each sample cuvette. The cuvettes were stoppered and shaken for 10 to 15 
seconds to quench the gentian violet in the test solutions. The 
spectrophotometer was zeroed again with the acid-alcohol blank and the 
absorbence of the quenched solutions was read. The O.D. of each of the 
quenched solutions was recorded. 
At any time, after passage of the extract solution through the 
chromatographic column, the solution appeared cloudy, the solution was 
warmed in a water bath for 1 to 2 minutes and the cloudiness disappeared. 
The absorbence of the sample solutions was identified by the following 
symbols: 
Sample Identity Absorbence Code 
Prequenched O.D. A.sub.x 
Quenched O.D. A.sub.q 
Calculation of gentian violet recovery from tissue samples is as follows: 
##EQU1## 
wherein: A.sub.x = absorbence at 590 m.mu. of unquenched solution. 
A.sub.q = absorbence at 590 m.mu. of quenched solution. 
A.sub.k = absorbence at 590 m.mu. of 1 ppm of gentian violet - acid alcohol 
standard solution. 
M.sub.1 = ml of acid alcohol used to extract gentian violet from the tissue 
samples (10 ml/gm of tissue sample). 
And: 
##EQU2## 
A sample calculation is: 
A.sub.x = 0.042 O.D. 
A.sub.q = 0.040 O.D. 
A.sub.k = 0.250 O.D. 
M.sub.1 = 50 ml of acid-alcohol. 
##EQU3## 
EXAMPLES 2 TO 24 
Example 1 was repeated 23 times using chicken liver samples from chickens 
which had been fed feed which contained gentian violet. (A number of 
chicken liver control samples, i.e., from chickens which had been fed feed 
which did not contain any gentian violet, were also run using the 
procedure of Example 1.) Livers have been found to have the highest 
gentian violet residues. 
The broiler chickens had been split into three groups. Group 1 (Examples 2 
to 9) was fed feed having 0 ppm of gentian violet. Group 2 (Examples 10 to 
16) was fed feed having 7 ppm of gentian violet. Group 3 (Examples 17 to 
24) was fed feed having 35 ppm of gentian violet. All of the chickens were 
fed for 56 days. The chickens of Examples 2, 3, 10, 11, 17 and 18 were 
sacrificed 4 hours after the final dose of gentian violet. The chickens of 
Examples 4, 5, 12, 19 and 20 were sacrificed 12 hours after the final dose 
of gentian violet. The chickens of Examples 6, 7, 13, 14, 21 and 22 were 
sacrificed 24 hours after the final dose of gentian violet. The chickens 
of Examples 8, 9, 15, 16, 23 and 24 were sacrificed 48 hours after the 
final dose of gentian violet. 
The control samples of Examples 2 to 9 and the samples of Examples 10 to 16 
were quenched with 0.01 ml of 0.26 percent sodium hypochlorite. The 
samples of Examples 17 to 24 were quenched with 0.05 ml of 0.26 percent 
sodium hypochlorite. The optical density of the 1 ppm HCl-ethanol standard 
repeatedly was 0.250. 
Table I 
______________________________________ 
Example Optical Quenched Opti- 
p.p.m. of 
Number Density cal Density Gentian Violet 
______________________________________ 
2 0.070 0.070 0.00 
0.075 0.075 0.00 
0.075 0.075 0.00 
3 0.048 0.048 0.00 
0.051 0.051 0.00 
0.048 0.048 0.00 
4 0.039 0.039 0.00 
0.039 0.039 0.00 
0.031 0.031 0.00 
5 0.047 0.047 0.00 
0.041 0.041 0.00 
0.045 0.045 0.00 
6 0.083 0.083 0.00 
0.080 0.080 0.00 
0.082 0.082 0.00 
7 0.078 0.078 0.00 
0.081 0.081 0.00 
0.089 0.089 0.00 
8 0.060 0.060 0.00 
0.060 0.060 0.00 
0.055 0.055 0.00 
9 0.060 0.060 0.00 
0.051 0.051 0.00 
0.060 0.060 0.00 
10 0.029 0.023 0.24 
0.035 0.028 0.28 
0.031 0.024 0.28 
11 0.031 0.024 0.28 
0.029 0.022 0.28 
0.031 0.024 0.28 
12 0.038 0.035 0.12 
0.051 0.048 0.12 
0.040 0.037 0.12 
13 0.095 0.093 0.08 
0.100 0.095 0.20 
0.090 0.087 0.12 
14 0.102 0.098 0.16 
0.103 0.102 0.04 
0.095 0.090 0.20 
15 0.053 0.051 0.08 
0.048 0.045 0.12 
0.061 0.058 0.12 
16 0.042 0.040 0.08 
0.051 0.048 0.12 
0.050 0.046 0.16 
17 0.060 0.041 0.84 
0.063 0.041 0.88 
18 0.073 0.056 0.068 
0.073 0.055 0.72 
19 0.051 0.040 0.44 
0.048 0.037 0.44 
20 0.028 0.017 0.44 
0.022 0.011 0.44 
21 0.041 0.025 0.64 
0.076 0.056 0.80 
22 0.068 0.053 0.60 
0.051 0.036 0.60 
23 0.047 0.038 0.36 
0.050 0.041 0.36 
24 0.081 0.075 0.24 
0.076 0.070 0.24 
______________________________________ 
Table I demonstrates that the quenching agent (NaOCl, in the amounts used, 
only quenches the gentian violet. This fact is demonstrated by the 
absorbence readings of the liver samples of the control birds (see Table 
I) in which no change of O.D. is found when the quenching solution is 
added to the control eluant. (The sensitivity of the analytical method, 
using the 13 mm cuvette, is such that 0.04 ppm gentian violet can be 
detected in the eluant. Use of the Beckman D.U. spectrophotometer equipped 
with the 100 mm cell, enables detection of gentian violet residues to 
approximately 0.005 ppm.) 
The results of Table I show that the gentian violet residue is lower with 
time after the last intake of feed which contains gentian violet. 
EXAMPLE 25 
Example 1 was repeated using a chicken liver control sample. The analysis 
was run without knowing a control sample was involved. The quenching was 
done with 0.01 ml of 0.26 percent sodium hypochlorite. The tissue analysis 
results for the control sample (four separate batches or runs) is: 
Table II 
______________________________________ 
Optical Quenched Opti- 
ppm of 
Density cal Density Gentian Violet 
______________________________________ 
0.065 0.065 0.00 
0.058 0.058 0.00 
0.070 0.070 0.00 
0.038 0.038 0.00 
______________________________________ 
EXAMPLE 26 
This example was run to determine that the liver was the target tissue 
(i.e., had the highest gentian violet residue), and that the quenching 
technique of this invention was accurate. The residual levels of gentian 
violet in parts per million (ug/g) in kidneys and livers taken from 
sacrificed young chickens after repeated oral administrations of .sup.14 
C-gentian violet was determined. 
The specific activity of the .sup.14 C-gentian violet (or crystal violet) 
was determined to be 5.8 .+-. 0.2 uCi/mg. (Another source determined the 
specific activity to be 5.99 uCi/mg.) 
Thirty-six animals, 7-day old male cockerels, were received and housed in a 
galvanized steel, wire-bottom rabbit cage. Water and chicken starter feed 
was provided ad libitum. Five days after being received, 33 chicks were 
selected and individually housed in galvanized steel, wire-bottom rat 
cages. 
An aqueous solution of .sup.14 C-gentian violet was prepared by dissolving 
50.4 mg of the compound in water and bringing the solution to volume in a 
100 ml volumetric flask. The lower dose level was prepared by diluting 20 
ml of the 0.5 mg/ml solution to 100 ml with distilled water. The second 
dose was referred to as the 1.times. dose level. The first solution was 
referred to as the 5.times. dose level. 
Each administration of each dose was performed by drawing the preparation 
into a 1 ml disposable Tuberculin Plastipak syringe fitted with a No. 20 
gavage needle; removing air bubbles from syringe and needle; filling the 
syringe to the appropriate volume; inserting the gavage needle into the 
animal's esophagus (covering the needle to the hub) and dispensing the 
preparation into the gastro-intestinal tract. Each chicken was weighed 
prior to dosing. If the animal weighed more than 100 gm, 0.25 or 0.05 mg 
.sup.14 C-gentian violet was administered. If the animal weighed less than 
100 gm, the appropriate dose level was prorated according to the animal's 
weight. Thus, an animal weighing less than 100 gm and receiving the 
5.times. dose received 2.5 mg .sup.14 C-gentian violet per kg; an animal 
weighing less than 100 gm and receiving the 1.times. dose received 0.5 mg 
.sup.14 C-gentian violet per kg. 
The preparations were administered on 12 consecutive days at 8:30 AM. The 
5.times. dose was administered to 20 birds, and the 1.times. dose was 
administered to 13 birds. The chickens were numbered from 1 to 33 and 
sacrificed at 4, 12, 24, 48 and 72 hours after administering the last 
dose. 
Each chicken to be sacrificed was anesthetized with ether and exsanguinated 
from the jugular vein using a syringe flushed with an aqueous solution of 
0.5 mg sodium heparin U.S.P./ml distilled water. After the animal died, 
the abdominal cavity was exposed and the duct between the liver and the 
gall bladder was tied off. The liver was then removed, cutting the bile 
duct between the liver and the knot. The kidneys were then removed. 
Both organs were weighed after being removed from the body. Immediately 
after weighing, duplicate aliquots of each organ were weighed into 
Combusto-Cones (Cat. No. 5065913; Packard Instrument Co.; Downers Grove, 
Illinois). Aliquots weighed between about 160 and 380 mg. The remainder of 
each organ was placed in a capped scintillation vial and frozen. 
All aliquots of organs were air-dried; the paper cones were tightly folded 
and samples were combusted in a Packard Model 305 Tri-Carb Sample 
Oxidizer. The .sup.14 C module of the oxidizer was calibrated to deliver 
methanol, ethanolamine, and scintillation cocktail in a ratio of 9:5:6 
into the scintillation vial. Performance checks (Manual 2118, Packard 
Instrument Company, pp. 208 to 2-10) were performed each day samples were 
combusted. A mean recovery of at least 95.0 percent and a memory of no 
more than 0.05 percent was required before samples were oxidized. A 
determination of oxidizer blank .sup.14 C trapping efficiency, and memory 
were also performed after oxidizing samples. .sup.14 C activity in samples 
prepared by oxidation was corrected for the oxidizer blank and the 
trapping efficiency. The scintillator for the oxidizer was 0.8 percent 
Preblend 2a60 in toluene (w/v). Preblend 2a60 is a preblended fluor 
composed of 91 percent PPO:2,5-diphenyloxazole and 9 percent 
POPOP:1,4-bis-2-(5-phenyloxazolyl)-benzene. 
All liquid scintillation determinations of .sup.14 C activity were made 
with a Searle Analytic Mark I Model 8725 liquid scintillation system. 
Counting efficiency for samples was determined by the external standard 
channels ratio method. The channel attentuator settings were: Channel A-A, 
870; Channel B-E, 760; and Channel C-B-O. Quench curves were plotted from 
the B/A ratio versus counting efficiencies of sealed standards containing 
a known amount of .sup.14 C activity and varying degrees of quenching. 
Sample counting efficiencies were found on the standard quench curve 
corresponding to the sample B/A counted with the external standard. All 
samples and blanks were counted for 10 minutes. 
Net cpm (counts per minute) for each sample was computed by subtracting the 
blank cpm from the sample's gross cpm. Net cpm was divided by 
scintillation counter efficiency to compute dpm (disintegrations per 
minute). Sample dpm were divided by oxidizer trapping efficiency to 
correct for oxidizer performance. Dpm/gram of sample was calculated by 
dividing corrected sample dpm by the sample aliquot weight (in grams). The 
concentration of ug .sup.14 C-gentian violet per gram of tissue (ppm) was 
computed by dividing the sample dpm/gm by 13.29 .times. 10.sup.6 dpm per 
mg .sup.14 C-gentian violet. This result was multiplied by 1000 to convert 
mg to ug, thereby giving ppm. Total dpm for an organ was computed by 
multiplying dpm/gram by the weight of the organ in grams. 
Eighteen aliquots (0.2 gm) were taken of a fresh liver from a 15-day-old 
chick, and each aliquot was weighed into a tared Combusto-Cone; the chick 
was exposed to no radioactive dose. Five sets of three aliquots were 
spiked, with 10, 50 and 100 ul Hamilton syringes, with varying amounts of 
an ethanolic solution of .sup.14 C-gentian violet to give concentrations 
of 1, 3.75, 7.5, 19 and 38 nanograms .sup.14 C-gentian violet per gram of 
tissue (ppb). The samples were air-dried at room temperature, oxidized, 
and counted for 10 minutes according to the procedures described above. 
The dpm per gram and nanograms per gram (ppb) were computed for each of 
the 18 samples. 
Table III sets out the mean residues of .sup.14 C-gentian violet in livers 
and kidneys for the 2.5 mg/kg (5.times.) and the 0.5 mg/kg (1.times.) dose 
levels. The amounts of .sup.14 C-gentian violet found in each individual 
tissue are set out in Table IV. The relative variation among samples taken 
at one time, as measured by the percentage of standard error of the means 
(percent S.E.M.), is greater for the livers than for the kidneys. An 
unusually high concentration of .sup.14 C-gentian violet was found in the 
liver of chick no. 31 sacrificed at seventy-two hours after administering 
the last dose. The high concentration of radioactivity can partially be 
explained by the low organ weight. Contamination from a ruptured bile duct 
is also possible; however, whenever the bile duct did burst during the 
necropsy, the liver was carefully washed with a small volume of distilled 
water. The kidney of this chick also had a high concentration of 
radioactivity. 
TABLE III 
______________________________________ 
Mean Tissue Residues of Gentian Violet 
After Oral Administrations of .sup.14 C-Gentian Violet 
(with Standard Errors of the Mean) 
5X Dose 1X Dose 
Post-Dose 
Liver Kidneys Liver Kidneys 
Time (ppm) (ppm) (ppm) (ppm) 
______________________________________ 
4 hrs. 0.779 0.477 0.284 0.102 
S.E.M. 0.084 0.078 0.075 0.018 
% S.E.M. 11 16 35 8 
12 hrs. 0.500 0.267 0.120 0.079 
S.E.M. 0.156 0.037 0.048 0.019 
% S.E.M. 31 14 23 24 
24 hrs. 0.679 0.210 0.114 0.067 
S.E.M. 0.162 0.036 0.051 0.022 
% S.E.M. 24 17 45 33 
48 hrs. 0.275 0.104 0.102 0.039 
S.E.M. 0.060 0.015 0.005 0.001 
% S.E.M. 22 14 5 3 
72 hrs. 0.686* 0.150* 0.034 0.024 
S.E.M. 0.021** 0.006** 0.007 0.002 
% S.E.M. 10 8 21 8 
______________________________________ 
Notes: 
*A sport, high value among three liver and kidney samples taken at this 
time. Mean of two liver values is 0.205. Kidney samples: 0.073. 
**S.E.M. based on two samples. 
Table IV 
______________________________________ 
.sup.14 C In Cockerel Organs Four Hours 
After Administration of .sup.14 C-Gentian Violet 
Organ 
Weight dpm/ dpm/ ug/gram 
Sample (gm) Organ gram (ppm) 
______________________________________ 
5X Dose 
Liver No. 
22 3.215 44,618 13,878 1.044 
15 5.133 50,607 9,859 0.742 
16 5.330 42,844 8,038 0.605 
17 4.834 46,652 9,651 0.726 
Kidneys 
No. 
22 1.216 11,116 9,183 0.691 
15 1.923 9,227 4,798 0.361 
16 1.875 8.867 4,729 0.356 
17 1.878 12,505 6,658 0.501 
1X Dose 
Liver No. 
1 7.070 21,804 3,084 0.232 
2 6.306 36,280 5,753 0.433 
3 6.548 16,323 2,493 0.188 
Kidneys 
No. 
1 2.395 2,836 1,184 0.089 
2 2.613 4,271 1,634 0.123 
3 1.790 2,272 1,269 0.095 
.sup.14 C in Cockeral Organs Twelve Hours 
After Administration of .sup.14 C-Gentian Violet 
Organ 
Weight dpm/ dpm/ ug/gram 
Sample (gm) Organ gram (ppm) 
______________________________________ 
5X Dose 
Liver No. 
18 5.630 66,438 11,801 0.888 
19 6.033 24,948 4,135 0.311 
20 6.170 17,753 3,201 0.241 
21 6.972 26,299 3,772 0.284 
23 6.611 68,033 10,291 0.774 
Kidneys 
No. 
18 2.274 11,939 5,250 0.395 
19 2.086 6,796 3,258 0.245 
20 2.136 6,537 3,060 0.230 
21 2.447 5,712 2,334 0.176 
23 2.027 7,815 3,855 0.290 
1X Dose 
Liver No. 
4 5.500 5,477 996 0.075 
5 6.357 5,889 927 0.070 
6 6.715 19,188 2,857 0.215 
Kidneys 
No. 
4 2.130 1,746 820 0.062 
5 2.172 1,696 764 0.057 
6 2.211 3,452 1,561 0.117 
.sup.14 C in Cockerel Organs Twenty-Four Hours 
After Administration of .sup.14 C-Gentian Violet 
Organ 
Weight dpm/ dpm/ ug/gram 
Sample (gm) Organ gram (ppm) 
______________________________________ 
5X Dose 
Liver No. 
24 6.289 60,300 9,588 0.721 
25 6.158 67,384 10,942 0.823 
26 5.351 68,033 12,714 0.957 
27 7.267 20,674 2,845 0.215 
Kidneys 
No. 
24 2.060 6,185 3,002 0.226 
25 2.176 6,443 2,961 0.223 
26 1.950 7,315 3,751 0.282 
27 2.476 3,623 1,463 0.110 
1X Dose 
Liver No. 
7 5.047 14,586 2,890 0.217 
8 6.222 5,165 830 0.062 
9 8.519 7,248 851 0.064 
Kidneys 
No. 
7 1.848 2,705 1,463 0.110 
8 2.318 1,481 639 0.048 
9 2.930 1,644 561 0.042 
.sup.14 C in Cockerel Organs Forty-Eight Hours 
After Administration of .sup.14 C-Gentian Violet 
Organ 
Weight dpm/ dpm/ ug/gram 
Sample (gm) Organ gram (ppm) 
______________________________________ 
5X Dose 
Liver No. 
28 5,788 15,334 2,649 0.199 
29 5.206 27,176 5,220 0.393 
30 4.390 13,617 3,102 0.233 
Kidneys 
No. 
28 2.237 2,197 982 0.074 
29 2.276 3,750 1,647 0.124 
30 1.911 2,882 1,508 0.113 
1X Dose 
Liver No. 
10 5.917 8,396 1,419 0.107 
11 4.701 6,073 1,292 0.097 
Kidneys 
No. 
10 2.321 1,186 511 0.038 
11 1.638 863 527 0.040 
.sup.14 C in Cockerel Organs Seventy-Two Hours 
After Administration of .sup.14 C-Gentian Violet 
Organ 
Weight dpm/ dpm/ ug/gram 
Sample (gm) Organ gram (ppm) 
______________________________________ 
5X Dose 
Liver No. 
31 2.079 45,587 21,927 1.650 
32 4.491 10,991 2,447 0.184 
33 4.428 13,245 2,991 0.225 
Kidneys 
No. 
31 1.236 4,998 4,044 0.304 
32 2.084 1,843 844 0.067 
33 1.720 1,792 1,042 0.078 
1X Dose 
Liver No. 
12 11.982 7,261 546 0.041 
13 10.383 3,744 361 0.027 
Kidneys 
No. 
12 2.916 972 333 0.025 
13 2.627 759 289 0.022 
______________________________________ 
When the data in Table III is plotted in a two-cycle semilogrithmic 
(Y-axis) scale), a plot of the best fit of log of concentration vs. time 
to a straight line can be achieved. The mean concentrations of gentian 
violet oscillate around the idealized elimination curves; in most cases up 
to 24 hours, the idealized curve passes within the range of the standard 
error of the mean. Table V presents the elimination rate constants and the 
half-lives of gentian violet computed from the linear regression curve of 
the data. The correlation coefficient from each linear regression is also 
given in Table V. 
The data indicate that gentian violet is concentrated about twice as much 
by the liver as by the kidneys. Comparing the results from the 5.times. 
and 1.times. doses indicates that the concentrating action of each organ 
decreases when the dose increases. At four hours after the last 
administration of the compound, there is 2.7 times as much radioactivity 
in the 5.times. livers as in the 1.times. livers; there is 4.7 times as 
much radioactivity in the 5.times. kidneys as in the 1.times. kidneys. The 
mean half-life of gentian violet in the two organs at dose levels is 32.2 
hours. The compound does not disappear more rapidly from one organ than 
the other. 
Table V 
______________________________________ 
Disappearance Rates of .sup.14 C-Gentian Violet 
From Livers and Kidneys 
Elimination 
Rate Constant Half-Life 
(K.sub.e) (t.sub.1/2) 
Correlation 
(hr.sup.-.sup.1) (hours) Coefficient 
______________________________________ 
5X Dose 
Livers -0.0193 35.9 0.935 
Kidneys -0.0262 26.7 0.974 
1X Dose 
Livers -0.0247 28.0 0.959 
Kidneys -0.0209 38.0 0.998 
______________________________________ 
A comparison of the chemical method residues (see Examples 2 to 24) to the 
C-14 residues demonstrates that the residue levels in livers from birds 
dosed "lifetime" with cold gentian violet very closely approximate the 
residue levels in the C-14 studies for all post-dose periods tested. 
Examination of the data in the C-14 study indicates the following tissue 
residues to be present at 72 hours post-dose: 
Sample Residue 72 hrs. post-dose 
______________________________________ 
Liver 0.034 ppm 
Kidney 0.024 ppm 
Skin/fat 0.016 ppm 
Breast muscle 
0.012 ppm 
Thigh muscle 0.012 ppm 
Gizzard muscle 
0.002 ppm 
Heart muscle 0.007 ppm 
______________________________________ 
It would appear from the close correlation of the chemical method residue 
results with the C-14 study that it could be expected, with a reasonable 
withdrawal time of 3 to 5 days, that the residue in the target tissue 
(liver) would be at or below the lowest detection capability of the 
chemical residue method (0.04 ppm) and would be approaching zero residue 
by 5 days withdrawal. 
EXAMPLE 27 
Example 26 was repeated using spiked tissues (chick) instead of tissue 
taken from chicks fed feed which contained gentian violet. The results of 
the experiments with spiked tissue samples are presented in Table VI. 
The 95 percent confidence limit was computed by multiplying the standard 
deviation by the value for Student's t-ratio (4.303) for the 95 percent 
confidence limit and 2.degree. of freedom. The mean dpm/gram for the 
unspiked samples falls within the 95 percent confidence range of the first 
two spike levels. This indicates that by the criteria of 95 percent 
confidence limits, the detection limit is above 4 ppb, i.e., 10 dpm. This 
result corresponds to a detection limit of twice the background, since the 
dpm/gm for 7.5 dpm is about two times the dpm/gm for unspiked tissue. 
Table VI 
__________________________________________________________________________ 
Detection of .sup.14 C-Gentian Violet in 
Spiked Liver Samples 
.+-. 95% 
Lower 
Volume 
Activity 
Mean Confidence 
Confidence 
of Added dpm/ Mean Limit Limit 
Standard 
dpm dpm/g 
Sample 
dpm/g (dpm/g) 
(dpm/g) 
__________________________________________________________________________ 
0 0 0 41.1 203 .+-. 
102 -- 
3.63 2.7 13.7 
55.4 283 .+-. 
196 87 
13.5 10 50.7 
65.5 331 .+-. 
136 195 
26.9 20 97 82.2 400 .+-. 
72 328 
67.3 50 266 114.8 
608 .+-. 
296 312 
134.6 
100 522 201.0 
1049 .+-. 
652 397 
__________________________________________________________________________ 
EXAMPLE 28 
In order to check the sensitivity of the methodology of this invention the 
instrumentation accuracy of instruments, etc., used in Examples 2 to 24, 
such was repeated using the chicken tissue spiked at the following gentian 
violet levels: 
0.1 ppm 
0.01 ppm 
0.02 ppm 
0.03 ppm 
0.04 ppm 
0.05 ppm 
0.005 ppm 
Sensitivity in the 13 mm cell was shown to be 0.04 ppm. 
Sensitivity in the 100 mm cell was shown to be 0.005 ppm.