Report
Good Laboratory Practices in Analytical Chemistry
William Horwitz
Bureau of Foods Food and Drug Administration Washington, D.C. 20204
Raymond P. Mariella, executive director of the American Chemical Society, in some excerpts published in Chemical & Engineering News (1) used a rather provocative title in another context, "A little internal honesty." I liked t h a t title, although only one small paragraph is directly appropriate to our concerns today. Dr. Mariella states, while discussing generalities about the nature and functions of chemists, "Because of our preoccupation with t r u t h we have difficulties with issues concerning halftruths. This issue doesn't stop politicians, lawyers, or government bureaucrats— who act on these matters frequently affecting us." This statement is a fine eyecatcher or earcatcher. Of course, it too is a halftruth. In our well-intentioned activities, we scientists, through oversight, negligence, and indifference, often cause our own problems. One of the problems which has surfaced relatively recently in some laboratories is the apparent gross mismanagement of scientific aspects of toxicological studies designed to establish the safety of our foods and drugs. You are painfully aware of the background which has led to the publication of the Food and Drug Administration's (FDA) proposed regulations on Good Laboratory Practice for Nonclinical Laboratory Studies (GLPs). T h e poor laboratory practices which were uncovered in animal testing laboratories were initially excused on the basis of a lack of standards and protocols for the conduct of toxicological and safety studies. But as soon as such This article not subject to U.S. Copyright Published 1978 American Chemical Society
standards and protocols were set forth, although admittedly in the legal format of the Federal Register of November 19,1976 (2), they were attacked as unnecessary, prohibitively expensive, and conducive to a stifling bureaucratic blanket on creative research and scientific investigations. I think we can simply disregard generalities and verbal sparring. Furthermore, although these regulations apply directly only to toxicological safety studies, there are sufficient requirements set forth in the proposed regulations dealing with identity, strength, quality, purity, stability, homogeneity, and protection from contamination and deterioration, t h a t the analytical chemist should realize t h a t he too is involved. His practices need review to ensure t h a t they correspond to sound scientific procedures. My thesis today is t h a t the application of good laboratory practices in analytical chemistry is merely a matter of common sense rather than bureaucratic high-handedness. Consider an FDA summary of some of the important problems encountered in the conduct of studies which have been submitted in support of the safety of certain drugs and food additives: "poorly conceived, carelessly executed, inaccurately analyzed or reported experiments "lack of awareness on the part of technical personnel of the importance of protocol adherence "inaccurate observations, record keeping and record transcriptions "failure of management to assure critical review of data or proper supervision of personnel ANALYTICAL CHEMISTRY, VOL. 50, NO. 6, MAY 1978 · 521 A
"use of poorly qualified or poorly trained personnel "disregard for proper laboratory, animal care, and data management procedures "failure to monitor studies performed in whole or in part by contract laboratories "lack of verification of the accuracy and completeness of scientific data "deliberate falsification of data by management and/or laboratory personnel."
experiment, the resulting observations, and the minor little points which invariably cropped up in the course of the work—like dropping the sample, the power going out, an instrument breaking down, etc. One of our chief chemists put it to his trainees this way, "When you fill out an analytical worksheet, think in terms of your having a fatal accident this afternoon. I will have to testify from your worksheet regarding everything that you had done and there is
One hundred of these animals will require about 2.5 tons of feed a year. Invoices should be available to show purchases of this order of magnitude. There are numerous simple, reasonable tests of this nature that enable any researcher or manager to verify that no gross blunder is occurring in such fundamentals as the feeding of the test substance and the controls. I hope one of our laboratory investigators will eventually compile a useful list of almost automatic checks on op-
no spiritualist available to institute extraterrestrial communication." The oversights in this area are astounding. The least understandable is when animals recorded as dead are resurrected at a later time; the final reports from some long-term toxicity studies show more animals at the end of the study than were present at the beginning. A simple inventory list of animals and their ultimate disposition would alert any reviewer to these types of errors. Observations recorded as raw data sometimes disappear from the final tables. A 1-lb bottle can never provide more than 1 lb of test substance; yet, this type of failure to account for material fed is frequent. An inventory of test substances must be maintained. If an animal consumes 2 oz of feed a day, it will eat nearly a pound a week or about 50 lb a year.
erating procedures which, if followed, will eliminate such gross blunders. In the analytical area, the laboratory record of the analytical work should reference the test substance by name, and by manufacturer's code and lot numbers as a minimum. When the bottle is empty, but not before, the label should be stripped, mounted, and stored. That label should also be dated as to receipt by facility and receipt by the laboratory; a number assigned for ease of data recording, access, and retrieval; and initials or names applied as a tracer, if needed. A portion of the material should be set aside in a "bank", properly identified, for reanalysis should any question ever arise as to its stability and suitability. Incidentally, many toxicological studies have failed to perform even the elementary analytical operations
Eliminate the one specific reference to "animal care", and every item on that list deserves examination by supervisors of analytical chemistry laboratories, not in the context of government regulations, but as a matter of "a little internal honesty". It has taken so long to examine this problem of unreliable data because for many years it was assumed that discordant results merely reflected biological variability. I recall pointing out some discontinuous data to the director of an eminent toxicological laboratory. Although he recognized the unusual pattern of the values, without a second thought he dismissed it with the observation, "That's what the animals showed." Another scientist, this time a foreign microbiologist, could not see the value of collaboratively testing the validity of the quantitative methods he used to develop and interpret data on the grounds that "the results are too variable".
Record Keeping If there is any single deficiency that permeates many of the laboratories which have been reviewed by FDA investigators, it is the matter of deficiencies in record keeping. Although my introduction to good laboratory practices is almost 40 years behind me, I still remember that the first thing impressed upon me, even in my high school science courses, was the importance of a good notebook. In general college chemistry and especially in analytical chemistry, we were instructed never to erase—always cross out incorrect entries and insert the correct ones, with an explanation for the error. I assume you remember receiving a warning about putting data on loose slips of paper, then disregarding the admonition, and learning first hand the importance of permanent records by having to repeat the experiment. Of course, some instructors emphasized this facet of laboratory work more than others. But anyone who saw his major professor only about once a week soon discovered for himself the importance of immediately recording the design of the
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of confirming the identity and purity of the material fed. Rather, they rely upon a label statement, which is not infallible. T h e protocol for the experiment should already have indicated how the test substances as well as positive and negative controls should be handled in the analytical laboratory. T h e procedure will vary depending upon whether it arrives in a single container or in multiple containers. Multiple containers may require compositing
your methods accordingly. However, watch out for a continuous series of negative or identical values. T h e y are just as likely to be indicative of an insensitive test as to indicate a real absence or the constant presence of an impurity or contaminant. Proper laboratory practices require t h a t you introduce a positive (spiked) sample occasionally so t h a t the system can respond when properly stimulated. It is much like the home smoke detector. Invite a smoker over
ÏK-PUJL^, or individual analyses. Similar considerations apply to the basic feed, b u t here the problem is selectivity. It is best to leave the choice of analyses to the nutritionists with regard to the primary nutrients (protein, carbohydrates, and fat) and secondary nutrients (vitamins and minerals), to be sure to analyze for those essential nutrients whose deficiency would confound the symptoms expected. Similarly, since it is impossible to analyze for all possible contaminants and additives, deliberate or inadvertent, let the toxicologists decide which compounds are likely to produce symptoms t h a t might imitate the expected response. T h e toxicologists should also be able to give an educated guess as to levels expected to result in biological response—so t h a t you can adjust the sensitivity and detectability of
occasionally so t h a t his habit can be used profitably to check t h a t your detector is in operating order. T h e subject of design of protocols is very complex. Consequently, it is often essential to design protocols almost on an individual experiment basis. T h e requirement for record keeping, however, is universal. Every observation or variable amenable to quantitation should be recorded, if only by a check mark to indicate t h a t it had been performed, like feeding the animals, or cleaning the cages. To gild the lily, you might even indicate the times of feeding and when the lights were turned on and off. This is not m e a n t to be facetious at all. Only recently have we discovered t h a t light affects the leaching of toxic cadmium from the pigments used to color eating utensils.
At this point we should say a word about methods of analysis. For many of the purposes of quality control, it is not necessary to utilize the most accurate and precise methods t h a t have been officially blessed by the Association of Official Analytical Chemists (AOAC) or by the Food and Drug Administration (FDA). In most cases you are merely interested in ensuring t h a t sufficient nutrients are present, t h a t contaminants are absent, and t h a t the test substance is present in the amount expected. All t h a t is needed are methods which provide the necessary information. Rapid screening methods are often sufficient for this purpose, b u t be sure t h a t the protocol provides for the occasional checking of a positive control to ensure t h a t the methods are doing the job they are intended to do. Photographs, charts, and recordings, and a sequential series of dated laboratory entries are the required evidence t h a t the tests have been performed. T h e supervisor (or in FDA terminology of the proposed G L P regulations, the study director) must assure himself t h a t the protocol is being followed and t h a t there is no deviation from the details of the methods chosen for the analyses. When protocols are changed for good and sufficient reason, the changes must be authorized and the reasons must be documented. When analytical methods are changed, the changes m u s t be validated to be sure there is no discontinuity in results. Changes should not be made a t the whim of the analyst or because he states, without evidence, t h a t the alteration is an improvement and it could not possibly influence the results. Scientists changing a protocol and analysts changing a method, without discussion and approval of the supervisor, and subsequent or concurrent documentation, are exhibiting more t h a n a little internal dishonesty. Another practice t h a t is frowned on is the dropping of analytical values, such as taking the best two out of three assays, particularly when recalculation using all values changes the conclusion from pass to fail.
Feed Homogeneity One important test t h a t is often overlooked is the necessity for checking t h a t the test substance has been incorporated uniformly into the feed. T h e operation of mixing seems so simple t h a t it has not been studied in detail from the point of view of achieving homogeneity. A recent publication from our laboratory (3) emphasizes the difficulty of incorporating parts per million of inorganic salts into an animal diet. Actually the operation was studied from the point of view of
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preparing a truly homogeneous analytical sample. If this small operation proved difficult, imagine t h e problems involved in increasing t h e size of t h e operation several orders of m a g n i t u d e . Failure to achieve homogeneous diets u n d o u b t e d l y is responsible for m u c h of w h a t is ascribed to "biological variability". You have seen the sawtooth t y p e of biological response as a function of test c o m p o u n d concentration. Can you imagine n a t u r e routinely operating in such a periodic fashion? Or is t h e r e a more reasonable explanation t h a t could be uncovered by routine application of analytical chemistry and, more i m p o r t a n t , by taking m a n a g e m e n t action to investigate such a p p a r e n t discrepancies or discontinuities?
Calibration and Standards A final area in which deficiencies are n u m e r o u s in laboratories reviewed by t h e FDA is the testing a n d calibration of e q u i p m e n t . We have become hypnotized by the ease of operation of m u c h of our e q u i p m e n t a n d gadgetry. As long as the pen moves, we ass u m e t h a t it is moving properly. Yet we often overlook t h e fact t h a t most of our i n s t r u m e n t s operate on a comparison principle—comparing t h e response of our unknown to t h e response of a similar s t a n d a r d . We often forget this principle when it is h i d d e n in t h e innards of our black boxes of single pan balances, spectrophotometers, and microcomputers. B u t even if we realize this principle, how often do we apply it? W h e n was t h e last time you p u t a s t a n d a r d weight on your balance? For quality control purposes, t h e reference material need not be a weight verified by t h e National B u r e a u of S t a n d a r d s . Any stable piece of m e t a l can be placed on the balance a t reasonable intervals. Keep a control c h a r t of t h e weight shown by t h e balance. W h e n t h e readings begin to deviate by more t h a n 2 s t a n d a r d deviations from the historical m e a n (regardless of its absolute mass if it is within the useful range of operations), an investigation is called for. Similarly, a piece of colored glass of the p r o p er thickness can produce a control c h a r t for spectrophotometers. T h i s can be a little tricky. Even t h e N a t i o n al B u r e a u of S t a n d a r d s h a d to warn its users of a slow deterioration in absorbance of one of its s t a n d a r d glasses used as an absorbance reference. Periodic preventive m a i n t e n a n c e of ins t r u m e n t s , keeping a log of their performance, and d o c u m e n t a t i o n of reference s t a n d a r d s are simply good laboratory practices. I have compiled a list of t h e common laboratory instrum e n t s , m a i n t e n a n c e frequency, a n d useful reference materials or tests t h a t are used in F D A field laboratories (4).
Calculations I learned long ago t h a t even t h o u g h I might think I am p r e t t y good in m e n t a l arithmetic, I j u s t should not t r u s t myself. I always a d d a column of figures twice—once down a n d once up. Even t h e a d v e n t of t h e h a n d calculator has not improved my reliability by m u c h , since I find myself occasionally punching the wrong key or not pressing it h a r d enough. If I can venture a guess on t h e two most costefficient good laboratory practice operations when something a p p e a r s to be wrong, they would be (1) checking calculations (by an i n d e p e n d e n t person; it's so easy to m a k e t h e same mist a k e twice); a n d (2) preparing new s t a n d a r d solutions (which is really t h e chemical equivalent of basic a r i t h m e tic operations) a n d a new s t a n d a r d curve. P e r h a p s you too have discovered t h a t a chemist will do almost a n y t h i n g to avoid preparing new s t a n d a r d solutions a n d calibration curves. H e will s p e n d hours tracing down a colleague who is r u m o r e d to have p r e p a r e d t h e required solution several years ago instead of spending the 15 min required to m a k e a new one. It is also good practice to compare t h e response of t h e new solution t o t h a t of t h e old one.
Housekeeping One other point deserves mentioning—the m a t t e r of good housekeeping. M a n y of the laboratories I have visited, including my own, are messy. Untidiness u n d o u b t e d l y cont r i b u t e s to t h e occasional u n t r a c e a b l e blunders a n d outliers which all analysts have encountered. We should keep our benches neater, our labels more legible, a n d our files more orderly. It is very difficult to exclude envir o n m e n t a l c o n t a m i n a n t s from our laboratories—cleansing agents, pest control agents, water t r e a t m e n t chemicals, a n d dust, t o n a m e only a few possibilities. T h e r e is no need t o deliberately introduce additional contamin a n t s by eating a n d smoking in t h e laboratory. T o reduce the possibility of errors a n d cross contamination, food, beverages, a n d snacks should n o t be stored in refrigerators dedicated to sample storage; personal belongings and personal medications should not be intermingled with reagents, medicated feed, positive controls, a n d similar necessities of t h e experiment.
Conclusion N o t h i n g t h a t I have m e n t i o n e d today smacks of bureaucratic nonsense. We have p e r h a p s grown lazy in becoming enticed by our m o d e r n laboratory i n s t r u m e n t a t i o n so t h a t we have neglected basic common sense
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a n d a little internal honesty in our operations: (1) K e e p records a d e q u a t e l y so t h a t it is obvious to anyone—our colleagues, supervisors, or a u d i t o r s — w h a t we have done, who did it, a n d when. (2) If we have promised to do a certain job in a specified m a n n e r , there is no excuse for not following those directions. Second guessing a n d short cuts require a separate validation a n d justification, which are usually more expensive t h a n following t h e original directions. (3) I n s t i t u t e proper m a i n t e n a n c e a n d calibration of i n s t r u m e n t s a n d monitor their performance constantly t h r o u g h t h e application of control c h a r t techniques on laboratory reference a n d s t a n d a r d materials.
References (1) R. P. Mariella, Chem. Eng. News, 55, 3 (Sept. 5, 1977). (2) Fed. Regist., 41 (225), 51206-30 (19 Nov. 1976). (3) K. W. Boyer and S. G. Capar, J. Toxicol. Environ. Health, 3, 747-55 (1977). (4) W. Horwitz, et al., "Analytical Food Chemistry," in "Quality Assurance Practices for Health Laboratories", S. L. Inhorn, Ed., Chap. 11, American Public Health Assoc, Washington, D.C., 1978. Delivered at the Symposium on Good Laboratory Practices, 17th Eastern Analytical Symposium, New York, N.Y., December 1,1977. This article is a chapter in a book of the same title to be published in spring 1978 by the Franklin Institute Press.
W i l l i a m H o r w i t z is d e p u t y associate director for science, B u r e a u of Foods, Food a n d Drug Administration, a n d is involved in the research and scientific aspects of the enforcement of t h e Food, Drug, a n d Cosmetic Act. H e is also executive director of t h e Association of Official Analytical Chemists a n d editor of t h e 1955, 1960, 1965, 1970, and 1975 editions of t h e Official M e t h o d s of Analysis of t h e Association of Official Analytical Chemists.
