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Bioavailability and Bioequivalence
339
4.3. Selection of GC/HPLC Column
It has been duly observed that based on the exact nature of the ‘analyte samples’, one may critically select
∑ normally-phase column and
∑ reverse-phase column.
1. Column Performances: Preferentially, all HPLC-columns must be purchased from the author-
ized suppliers and their respective performances need to be carried out according to the certifi cate
provided by the manufacturer or as per the standard operating procedure (SOP) of the ‘Bioanalytic
Laboratory’.
S. No. HPLC LC-MS GC-MS
8 To develop a feasible method for
combination of analytes in a
single run is rather diffi cult.
It is a lot easier to accom-
plish.
It is found to be easier when
the analytes do have the same
nature.
9 No specifi c gases are needed. Gases like N
2
, zero air, com-
pressed air are required.
He gas is only required.
10 Sample injection volume ranges
between 20 and 100 μL.
Sample injection volume
should be <40 μL.
Sample injection volume must be
<5 μL.
11 Needs minimum skill and training. Requires specialized expertise. Needs a special expertise.
12 On-line sample processing and
injection not that benefi cial.
On-line sample preparation
and injection enhances
throughput.
For certain specifi c molecules
solid-phase microextraction is
available adequately.
These essentially comprise
Normal Phase Columns e.g., silica, amino, CN etc.
Reverse Phase Columns e.g., C-18, C-8, C-4 etc.
The most commonly employed HPLC columns in HPLC, LC-MS and GC-MS are stated in
Table 5.5 together with their length, width, particle size and theoretical plates.
Examples
Table 5.5 Column dimensions of HPLC, LC-MS and GC-MS with regard to length, width, particle size
and theoretical plates
S. No. Instrument Length (mm) Width (mm)
Particle size (m)
Theoretical plates
(per m)
1 HPLC 150–300 4–4.6 5–10 50,000
2 LC-MS 50–75 2–4.6 3 –
3 GC 50 m 0.25 0.25 3,000 (capillary)
Essentials of Biopharmaceutics and Pharmacokinetics
340
2. Salient Features
These essentially include the following:
(a) Mostly one may observe that the retention time and length of column are found to be inversely
proportional.
(b) Length of column on being enhanced leads to improved
∑ selectivity and
∑ resolution
whereas both sensitivity and peak-shape may get reduced appreciably.
(c) Reduction in particle size of the adsorbent may give rise to better sharp peak shape, but it may
lower the life-span of the HPLC column signifi cantly.
(d) To develop a reasonably good HPLC method, one should be selective in choosing
∑ right type of column (viz. C-18, C-8 C-4) and
∑ appropriate dimensions.
(e) Use of ‘C-loaded columns’ may be used rather than unnecessarily increasing the length of the
column for handling the assays related to complicated analyte samples.
(f) To prevent damage caused to the main HPLC analytical column, one should always make use
of the ‘Guard Columns’.
3. Special Care of HPLC Columns: These predominantly consist of the following:
(a) All HPLC columns must be stored after proper washing as recommended/certifi ed by the
manufacturer.
(b) For the reverse phase HPLC columns it is always advised to do the necessary thorough wash-
ings with 100% water so as to get rid of the buffers that are left behind in the column
49
and
then with the requisite organic solvent(s) in the reverse order to remove the entrapped water,
if not, it would lead to serious ‘fungal growth’ inside the column.
(c) When the HPLC columns are not in use, it is required to be capped properly soon after the
washing operation.
4. Procedure for Regeneration of HPLC Column: Obviously, the laid down procedure the
regeneration of HPLC column do vary from the exact type of column, such as:
(a) Silica Columns: For the regeneration of silica columns in HPLC, it is required to pump in
nearly 50 mL of the following HPLC Grade solvents at the rate of 1–2 mL per min sequentially:
∑ Tetrahydrofuran (THF),
∑ Methanol,
∑ Aqueous acetic acid (2–4%),
∑ Aqueous pyridine (2–4%),
∑ Tetrahydrofuran and
∑ tert-2-Butylmethyl ether.
(b) Reverse Phase Columns: For the regeneration of the reverse phase columns in HPLC, it is
most preferred to pump in approximately 50 mL of the following HPLC grade solvents at the
rate of 1–2 mL per min sequentially:
∑ Water + 4 × 100 μL of DMSO (dimethyl sulphoxide) injected,
∑ Methanol,
∑ Chloroform and
∑ Methanol.
49
Otherwise, the salts in ‘buffer’ may crystallize and cause clogging of column.
Bioavailability and Bioequivalence
341
4.3.1. pH of Mobile-Phase Buffer
It is now required to select a proper buffer for HPLC, since its molarity and pH do play a prominent
and critical role in terms of better selectivity, sensitivity and resolution. However, the ideal pH range
essentially needed for the said mobile-phase buffers range between 2.5 and 7.5 solely depending upon
the very inherent nature of the ‘analyte sample’ being investigated. Nevertheless, in most instances, par-
ticularly in RP-HPLC, a decrease in pH invariably causes
∑ decrease in ‘retention-time’ and
∑ increase the ‘shape of peak’,
whereas, it would affect simultaneously ‘selectivity’ and ‘resolution’.
Important Points These include predominantly the following aspects:
1. It is always better to make use of 1–20 milli-molar buffer.
2. Evidently, an increase in the molarity of the buffer may no doubt afford a relatively good separa-
tion, but at the same time shall reduce
∑ shape of the peak,
∑ sensitivity and
∑ life of column.
3. Conventional Buffers for HPLC: The most abundantly used conventional buffers for HPLC
are as stated below:
∑ Sodium acetate,
∑ Sodium dihydrogen phosphate,
∑ Potassium dihydrogen phosphate,
∑ Ammonium dihydrogen phosphate and
∑ Tris-Buffers.
4. Ion Pairing Agents: Invariably, along with the aforesaid buffers [in (3)] it is advisable to make use
of the ion-pairing agents, such as butane, pentane, heptane, sulphonic acids, in different propor-
tions so as to sustain and retain the ionizable analyte samples upon the typical RP-HPLC columns.
5. LC-MS: The LC-MS hyphenated analytical technique a host of volatile buffers are used, such as:
∑ Acetic acid (0.1%),
∑ Formic acid (0.1%),
∑ Ammonium acetate and
∑ Ammonium formate.
Note
It is pertinent state here that one may obtain better results by carefully adjusting the pH of the
buffer with conjugated acid or base as per the necessary requirement.
6. Preparation of Buffers for HPLC: The various steps involved are as follows:
∑ It is usually prepared by using ‘Ultra-Sonicators’ for rapid dissolution of inorganic salts.
∑ Degassing of the resulting solution is a must.
∑ The degassed buffer solution is fi ltered via 0.24 μ membrane fi lter.
∑ Filtered HPLC-buffers are normally stored in ‘glass containers’ to avoid fungal growth.
∑ Prepared buffer solutions must be used within 48 hrs.
Essentials of Biopharmaceutics and Pharmacokinetics
342
4.3.2. Specific Composition of ‘Mobile-Phase’
In actual practice, the exact composition of the ‘mobile-phase’ exclusively rests upon the specifi c type
of such criteria as
∑ matrix,
∑ instrument,
∑ column selected and
∑ total number of ‘analyte sample’ being assayed.
LC-MS vs. HPLC: Obviously, in the particular instance of LC-MS, the precise and exact proportion
of the organic solvent needed usually remains very high as compared to the HPLC method. Besides, it
has been observed in many cases that the critical enhancement in the proportion of the organic solvent
directly increases the corresponding peak height/shape as well as sensitivity, whereas, it may minimize
the extent of both selectivity and resolution criteria simultaneously.
Development of HPLC Method: In the course of developing a new HPLC method it is always
advisable to allow the proper mixing of the buffer and solvent together in large quantum unless one
makes use of the quaternary HPLCsystem (which prevents wastage of solvent appreciably).
Use of Allegation Method: In has also been recommended to make use of the allegation method
intelligently for the development of HPLC method.
4.3.3. Column-Oven Temperature
The effective use of an optimized column-oven temperature is found to yield reasonably good repro-
ducibility in the retention time of the analyte sample.
Salient Features: These essentially comprise
1. Certain typical analyte samples do require higher temperature for the desired elution just above
the ambient temperature.
In a situation, when one has duly prepared
∑ solvent (50% v/v) and
∑ buffer (50% v/v)
and one intends to enhance the solvent ratio to 65% simply follow the underlying procedure of allegation:
5HTXLUHG
3XUH6ROYHQW 6ROYHQW
([LVWLQJ0RELOH3KDVH
([LVWLQJ0RELOH
3KDVH
Example
Note
The aforesaid ‘allegation method’ is solely meant for quantitative purposes; however, during fi nali-
zation of the method, the mobile phase has to be prepared afresh as and when needed.
Bioavailability and Bioequivalence
343
2. An increase in column temperature may help in the defi nite reduction in the retention time of
the analyte, but contrarily enhances the shape of the peak. Ultimately, it may affect upon both the
selectivity and resolution.
3. A signifi cant decrease in the viscosity of the mobile phase may be observed while increasing the
column temperature. Therefore, it usually indulges in accomplishing two glaring aspects, namely:
∑ permits enhanced fl ow rates and
∑ increases the diffusibility,
which predominantly reduces the critical resistance cause to the respective ‘mobile-phase mass
transfer’ largely.
50
Demerits of Increased Column Temperature: Following are the two most vital demerits
of increased column temperature in HPLC assays:
∑ possibility of analyte components and solvent components to undergo decomposition and
∑ Changes of vapour pressure of solvent(s) to rise, thereby enhancing the high-risk of bubbles
present in the detector that would ultimately give rise to the production of
_ an uneven baseline and
_ ghost (false) peaks.
4.3.4. Sample Injection Volume
Based on various experimental data, it has been duly established that the precise sample injection volume
fails to cause a noticeable effect upon the following aspects:
∑ selectivity,
∑ sensitivity,
∑ shape of peak and
∑ column life.
Importantly, an increase in sample injection volume may also enhance the sensitivity whereas, it would
reduce the shape of peak and column life signifi cantly. Therefore, it is mostly preferred to reduce the
sample injection volume till a situation demands it urgently.
Note
In some instances, an increase in the sample injection volume shall tend to merge even the impu-
rities right into the ‘analyte peak’.
4.3.5. Extra Column Volume
Interestingly, the ensuing volumes occurring within an HPLC equipment that eventually causes the
separation of the components present in the analyte sample are invariably termed as the extra column vol-
umes. It is worthwhile to state at this point in time that such ‘critical volumes’ that are strategically located
∑ just prior to the injector or
∑ just behind the detector
must not be regarded as the extra column volumes.
50
That is, the analyte molecules get diffused via the mobile phase faster at elevated temperatures.
Essentials of Biopharmaceutics and Pharmacokinetics
344
Necessarily, the extra column volumes must be maintained as low as feasible and possible, since they
could fi nally affect noticeably:
∑ separation of components and
∑ shape of peak.
In fact, the extra column volume is solely responsible for affording
∑ band broadening and
∑ band tailing.
4.4. Method Validation
After having developed an elaborated bioanalytical method it is very much desired and advisable to carry
out a ‘trial’ comprising a calibration curve. Besides, necessary ‘quality control’ checks has got to be done
before commencing a complete validation method.
In a broader perspective, the bioanalytical method validation essentially includes practically all of the
procedures which clearly prove and demonstrate that a specifi c method employed for the ‘quantitative
measurement of analyte samples’ in a given matrix, for instance, serum, plasma, blood and wine, is estab-
lished to be absolutely:
∑ reliable and
∑ reproducible,
for all future intended usage.
In general, the fundamental experimental parameters required predominantly for this ‘validation’
necessarily must include the following cardinal aspects, namely:
∑ selectivity,
∑ sensitivity,
∑ ruggedness and robustness,
∑ repeatability,
∑ accuracy,
∑ recovery,
∑ linearity and
∑ stability.
51
Suggested Readings
ASTM-Methods, Vol. 14.02 E 898–88, June 1990.
ASTM-Methods, Vol. 14.03 E 77–89, June 1990.
AST-Methods, Vol. 14.02 E 542–94, June 1990.
Blanchard J et al. Principles and Perspectives in Drug Bioavailability. New York: S. Karger, 1979.
Bourne DWA, Dittert LW. Pharmacokinetics, In: Banker GS, Rhodes CT, Eds. Modern Pharmaceutics, 2nd edn. New
York: Marcel Dekker, 1990.
51
It essentially comprises in-injector or post-processing, bench-top, freeze-thaw, short-term/long-term prepared
‘Stock Solutions’, matrix samples long term, and dilution integrity.
Bioavailability and Bioequivalence
345
Gibson CG, Skett P. Introduction to Drug Metabolism. London, UK: Chapman and Hall, 1986.
Hoener BA, Benet LZ. Factors infl uencing drug absorption and drug availability. In: Banker GS, Rhodes CT, Eds.
Modern Pharmaceutics. New York: Marcel Dekker Inc., 1990.
Mc Elnag IC. Buccal absorption of drugs. In: Swarbrick J et al. Eds. Encyclopedia of Pharmaceutical Technology, Vol. 1.
New York: Marcel Dekker Inc., 2007.
Meyer MC. Bioavailability of drugs and bioequivalence. In: Swarbrick J et al. Eds. Encyclopedia of Pharmaceutical
Technology, Vol. 1. New York: Marcel Dekker Inc., 2007.
Ritschel WA, Handbook of Clinical Pharmacokinetics, 3rd edn. Hamilton, IL: Drug Intelligence Publication, Inc., 1986
Rowland M, Tozen TN. Clinical Pharmacokinetics: Concepts and Applications, 3rd edn. New York: PA: BI Waverly Pvt.
LTD, 1996.
Shargel L et al. Applied Biopharmaceutics and Pharmacokinetics, 5th edn. Sydney, Australia: McGraw Hill, 2005.
Sloan KB. Prodrugs: Topical and Ocular Drug Delivery. New York: Marcel Dekker Inc., 1992.
Stockley IH. Drug Interactions: A Sourcebook of Drug Interactions, Their Mechanisms, Clinical Importance and Management,
2nd edn. London, UK: Blackwell Scientifi c Publications, 1991.
Synder LR, Stadalius MA. High Performance Liquid Chromatography: Advances and Perspectives, Horvath CS, Ed. New
York: Academic press, 1986.
United States Pharmacopoeia 27 and NF-22, 2008.
US-FDA: Guidelines for Industry-Bioanalytical Method Validation, 1992.
Welling PG et al. Pharmaceutical Bioequivalence. New York: Marcell Dekker Inc., 1991.
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Index
A
a1-acid glycoprotein or albumin 129
a1-acid glycoprotein 124, 125
a1-AGP or AAG 125
a1-globulins 131
a2-globulins 131
Abbreviated New Drug Application (ANDA) 249
Absolute Bioavailability 14, 292
Absolute Bioavailable Dosage 268
absorbed dose 228
absorption of drug 13
absorption pattern 28
Absorption Phase 21, 157
absorption rate constant 154
absorption via a fi rst-order rate process 160
Absorption 26
Accounting for Deviation of Michaelis–Menten
Equation 196
Accumulation Factor 227
Accumulation Index 227
Accumulation of Drug in Multiple Dosing 225
active drug product 27, 220, 296, 331
Active pharmaceutical ingredient 28
Active Transport 36, 66
Active Tubular Secretion (ATS) 176
Acyclovir 316
ADME 21, 123
Adrenocortical Hormone 33
Advantageous Interactions 276
Advantages of Latin-Square Design 263
age factor 80
Ageing phenomenon 218
Aims and Objectives of Bioavailability
Studies 251
Alkaline phosphatase 267
Almost complete miscibility 322
Amycolatopsis orientalis 141
analgesiometer activity cage, rota-rod apparatus 6
Analysis of Variance [ANOVA] 264
Analytical Methods 332
anatomic variance 48
Antianginal Drugs 209
Antiarrythmics 208
Antibiotics 212
Anti-frictional Agents 114
Antihypertensives 207
anti-neoplastic chemotherapy 279
Antineoplastic Agents 211
Applications of IVIVC 32
Applied Compression Force 109, 111
Aquaporins 46
Aqueous Solubility of Drug 238
aqueous solubility 70
Area Under Blood/Plasma Time Concentration
Curve (AUC) 252, 254
Area Under Curve Fluctuation (AUCF) 257
Area Under the Curve (AUC) 15, 252, 307
area under the plasma concentration–time curve
(AUC) 310
Assay of Biotransformation Product 332
Astrocytes 38
AUC for a Mono-Exponential Concentration Time
Course 273
AUC Value Estimated from Reference Plot 270, 271
AUC value for an Exponential Concentration Time
Course 270, 272
AUC Value for Extravascular Route 270
augmentation of ‘wetting’ 318
Average rate of drug absorption 21
average steady-state plasma–drug concentration 229
B
b-adrenergic agonist 4
b-Blockers 40
Index
348
b-Globulins 131
Balanced Incomplete Block Design (BIBD) 263
band broadening 344
band tailing 344
basket-type dissolution apparatus 302
Batch-Control Results 331
benefi t-to-risk ratio 10
biexponential drug–plasma concentration–time 272
biexponential equation 144
Biliary Excretion 189
Biliary Function 192
Binders and Granulating Agents 112
Binding of Drugs 125
Bioanalytical Instruments 337
Bioanalytical Method Development and Validation
for Bioavailability 336
Bioanalytical Methods 332
Bioavailability Studies as per the Helsinki
Declaration 297
Bioavailability Studies in Human 296
bioavailability studies 298
Bioavailability 6, 13, 30, 285, 286, 293, 298
bioavailable dose 269
bioavailable fraction 269
biochemical reaction 12
Bioequivalance Assessment and Data Evaluation
(or Importance of Bioequivalence) 293
Bioequivalence Assessment 293
Bioequivalence Data: Statistical Interpretation 263
bioequivalence evaluation studies 18
bioequivalence evaluation 306
bioequivalence problems 18
Bioequivalence Requirement 286
Bioequivalence Studies: Not Necessary 268
Bioequivalence Study Design 259
Bioequivalence Study Parameters 251
Bioequivalence Study Protocol 258
Bioequivalence 17, 327
Bioequivalence: The Canadian Regularly
Perspective 17
Bioequivalent Dosage Forms 286
Bioequivalents 251
biological half-life 185
Biopharmaceutic Considerations 73
Biopharmaceutics Classifi cation System (BCS) 29
Biopharmaceutics 3, 4, 6, 25
biotransformation phenomenon 13
biotransformation 20, 26
Blood Cerebrospinal Fluid Barrier
(BCSFB) 33, 39
Blood Flow 239
blood proteins 125
Blood sugar 266
Blood 173
Blood–brain barrier (BBB) 33, 36
Blood–Serum–Plasma 137
Blood–Testis Barrier (BTB) 33, 42
Body Surface Area (BSA) Rule 221
Bolus 140
Brand Name 287
Branded Drug Product 286
Branded Product 331
Buffered ‘aspirin tablets’ 319
Buffers 116
C
Capillary Endothelial Barrier (CEB) 33
Capillary Endothelial Cells 36
capillary walls 39
Captopril 207
Carbonic Acid (H
2
CO
3
) 133
Carbonic Anhydrase 132, 133
cardiac bypass surgery (CBS) 243
cardiac output 237
cardiovascular tissues 124
carrier-mediated absorption process 66
carrier-mediated processes 64
carrier-mediated transport process 65
Carr ier-Mediated Transport 63
Cartesian plot 155
Caternary Model 137, 147
Cell Membrane Barrier (CMB) 33–35
cell membrane components 50
Cell Membrane Structure 45
Central Compartment 141–144
Change in pH of Drugs
Microenvironment 319
Changes in GI Motility 277
Characteristic Features of Carrier-Mediated
Transport 64
Characteristics of Excipients 111
Chelation 276
chlorothiazide (CTZ) 133
chlorpromazine (CPZ) 133
Choroid plexus 39
choroidal cells 39
chylomicrons 130
circulatory functions 331
Clark’s Rule 221, 237
clinical investigative trials 298