In a series of posts, we are going to talk about method validation.

1. Part 1: Introduction-Is it valid, invalid or non-validated?
2. Part 2: What is method validation?
3. Part 3: Can we use some­one else’s val­i­dated method?
4. Part 4: What trig­gers ver­i­fi­ca­tion, re-validation or out right new val­i­da­tion of a method?
5. Part 5: What are the essen­tial terms in method validation?
6. Part 6: What is qual­ity assur­ance and qual­ity control?

Accord­ing to Dr. Lud­wig Huber in his book Val­i­da­tion and Qual­i­fi­ca­tion in Ana­lyt­i­cal Laboratories

Method val­i­da­tion is the process used to con­firm that the ana­lyt­i­cal pro­ce­dure employed for a spe­cific test is suit­able for its intended use. Results from method val­i­da­tion can be used to judge the qual­ity, reli­a­bil­ity and con­sis­tency of ana­lyt­i­cal results; it is an inte­gral part of any good ana­lyt­i­cal practice.

The United States Phar­ma­copeia (USP) is a non–governmental, offi­cial pub­lic standards–setting author­ity for pre­scrip­tion and over–the–counter med­i­cines and other health­care prod­ucts man­u­fac­tured or sold in the United States. The USP also sets widely rec­og­nized stan­dards for food ingre­di­ents and dietary sup­ple­ments. The USP sets stan­dards for the qual­ity, purity, strength, and con­sis­tency of these products–critical to the pub­lic health. The USP’s stan­dards are rec­og­nized and used in more than 130 coun­tries around the globe. These stan­dards have helped to ensure pub­lic health through­out the world for close to 200 years.

The USP has pub­lished spe­cific guide­lines for method val­i­da­tion for com­pound eval­u­a­tion. USP defines eight steps for validation:

1. Accu­racy (Bias)
2. Pre­ci­sion
3. Speci­ficity
4. Limit of detection
5. Limit of quantitation
6. Lin­ear­ity and range
7. Rugged­ness
8. Robust­ness

Put basi­cally and sim­ply, a val­i­dated method means that there has been some sort of rig­or­ous method of test­ing of the instruc­tions of the assay and its cal­i­bra­tion pro­ce­dure to pro­duce data that shows and proves that the method is suit­able for its intended purpose.

It might be use­ful to use a con­crete exam­ple so we can trans­lates the words and the con­cepts into the demon­stra­ble con­cept. In Head­space analy­sis by Gas Chro­matog­ra­phy with Flame Ion­iza­tion Detec­tor (HS-GC-FID) for EtOH, the intended pur­pose is to:

1. cor­rectly and uniquely iden­tify the tar­get analyte—ethanol—to the exclu­sion of every­thing else (that may likely be in the matrix—the blood—or in the envi­ron­ment of the col­lec­tion, stor­age, trans­porta­tion, sam­ple prepa­ra­tion, or analysis—contamination) and there­fore be spe­cific in its qual­i­ta­tive measure.
2. cor­rectly and uniquely iden­tify the quan­tity of the tar­get analyte—ethanol—to the exclu­sion of all other pos­si­ble inter­fer­ing com­pounds, and there­fore have a spe­cific mea­sure in terms of the quan­ti­ta­tive measurement.

You may be think­ing to your­self, ” Great. But as prac­tic­ing attor­neys, why should we care?”

With­out a valid method, we can never achieve a val­i­dated result. If there is devi­a­tion, this will result in a non-validated result.

A good anal­ogy is that of a foundation.

Think of method val­i­da­tion as mak­ing a foundation

Of course, how you lay the foun­da­tion is going to affect the qual­ity and the dura­bil­ity of the build­ing it rests upon. If the foun­da­tion is poor, it doesn’t mat­ter how per­fect the fram­ing, how per­fect the roof, how per­fect the blue board or dry­wall, and how per­fect the inte­rior sup­ports are in the sub­se­quent build­ing the house, the build­ing is in jeop­ardy and will even­tu­ally collapse.

Poor foun­da­tion means for disaster

Just as in lay­ing the cor­rect con­crete foun­da­tion, there are cer­tain cor­rect ways of doing it such as:

1. Deter­mine how thick the slab will be. This will be deter­mined by the use and size.
2. Exca­vate or dig down twice the thick­ness of the slab. If the con­crete foun­da­tion is going to be 3 inches, then you need to go down 6 inches.
3. Remove any large stones or garbage from the area you exca­vated, then rent a com­pactor and com­pact the ground.
4. Put down sand or gravel or some­thing for drainage so water will not come up through the slab.
5. Lay down a vapor bar­rier, espe­cially if the con­crete foun­da­tion is going to be used for inte­rior use such as a house.
6. Build a wood frame to lay a con­crete foun­da­tion. Make screed rails from 2-by-4 inch boards and put them on oppo­site sides around the out­side of the foun­da­tion. Span the screed rails with a straight­edge. This slides across the rails, remov­ing excess con­crete and help­ing to make the con­crete level.
7. Pour the con­crete for the foun­da­tion and allow the straight­edge to slide, lev­el­ing the con­crete as it goes. Once the con­crete is firm enough, remove the straight­edge and rails.
8. Fin­ish the con­crete foun­da­tion you are lay­ing with hand or power trow­els. Remove any imper­fec­tions the straight­edge has left, then let the con­crete foun­da­tion sit and cure.

so there are the cor­rect ways of per­form­ing method val­i­da­tion. How­ever, there is no “one way” to design and per­form val­i­da­tion exper­i­ments. As Dr. Huber wrote in his book, based on his expe­ri­ence, for exam­ple in try­ing to develop a liq­uid chro­mato­graphic method, the fol­low­ing sequence has proven to be use­ful in terms what exper­i­ments to run and in what order:

1. Selec­tiv­ity of stan­dards (opti­miz­ing sep­a­ra­tion and detec­tion of stan­dard mix­tures if selec­tiv­ity is insufficient)
2. Lin­ear­ity, limit of quan­ti­ta­tion, limit of detec­tion, and range
3. Repeata­bil­ity (short-term pre­ci­sion) of reten­tion times and peak areas
4. Inter­me­di­ate precision
5. Selec­tiv­ity with real samples
6. Trueness/accuracy at dif­fer­ent concentrations
7. Rugged­ness (inter­lab­o­ra­tory studies)

Accord­ing to Dr. Huber:

Once the method has been devel­oped and val­i­dated, a val­i­da­tion report should be pre­pared that includes the following:

• Objec­tive and scope of the method (applic­a­bil­ity, type).
• Sum­mary of methodology.
• Type of com­pounds and matrix.
• All chem­i­cals, reagents, ref­er­ence stan­dards, QC sam­ples with purity, grade, their source or detailed instruc­tions on their preparation.
• Pro­ce­dures for qual­ity checks of stan­dards and chem­i­cals used.
• Safety pre­cau­tions.
• A plan and pro­ce­dure for method imple­men­ta­tion from the method devel­op­ment lab to rou­tine analysis.
• Method para­me­ters.
• Crit­i­cal para­me­ters taken from robust­ness testing.
• List­ing of equip­ment and its func­tional and per­for­mance require­ments, e.g., cell dimen­sions, base­line noise and col­umn tem­per­a­ture range. For com­plex equip­ment, a pic­ture or schematic dia­gram may be useful.
• Detailed con­di­tions on how the exper­i­ments were con­ducted, includ­ing sam­ple prepa­ra­tion. The report must be detailed enough to ensure that it can be repro­duced by a com­pe­tent tech­ni­cian with com­pa­ra­ble equipment.
• Sta­tis­ti­cal pro­ce­dures and rep­re­sen­ta­tive calculations.
• Pro­ce­dures for QC in rou­tine analy­ses, e.g., sys­tem suit­abil­ity tests.
• Rep­re­sen­ta­tive plots, e.g., chro­matograms, spec­tra and cal­i­bra­tion curves.
• Method accep­tance limit per­for­mance data.
• The expected uncer­tainty of mea­sure­ment results.
• Cri­te­ria for revalidation.
• The person(s) who devel­oped and val­i­dated the method.
• Ref­er­ences (if any).
• Sum­mary and conclusions.
• Approval with names, titles, date and sig­na­ture of those respon­si­ble for the review and approval of the ana­lyt­i­cal test procedure.