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					 Welcome to Class!
        Kevin Olsen
    359 Richardson Hall
       973-655-4076
OlsenK@Mail.Montclair.Edu




                            1
 Class Expectations and Grading
• Grades will be awarded based on laboratory
  reports and homework assignments. There may be
  quizzes given in class depending on time
  limitations.
• Some homework assignments will require an oral
  presentation to the class.
• Unless specifically stated otherwise, the standards
  of documentation required on laboratory reports
  will be those found in a GMP / GLP regulated
  environment.
                                                    2
            Laboratory Reports
• All paperwork is a permanent record.
  Nothing will be discarded.
• All chromatograms will include:
1.   Student name
2.   Date
3.   Sample ID
4.   Chromatographic conditions
5.   System ID number and calibration dates

                                              3
 Laboratory Reports (continued)
• Laboratory reports will be checked by a
  second person before being turned in. The
  person checking the report will sign it.
• Any cross outs will consist of a single line,
  initials and date. In some instances such as
  a faulty injection, an explanation for the
  cross out will be included.
• No records will be kept in pencil.

                                                  4
 Laboratory Reports (continued)
• The identity and calibration data of each
  and every instrument used will be included
  on the laboratory report, this includes
  balances and pH meters. If an instrument is
  not calibrated, that fact will be noted.
• The manufacturer and lot number of each
  reagent used will be recorded. (Mobile
  phases are exempt from this rule.)
                                                5
The Cardinal Rules of GMP / GLP
• If it was not documented, it did not happen.
• To err is human, to destroy the evidence is a
  felony.
• Maintain every record as if it were going to
  be reviewed by the FDA.
• Your signature is precious, never sign,
  approve, or authorize anything you are not
  absolutely sure is correct.
                                              6
              Lesson 1
• Review of HPLC components
• Operating your HPLC




                              7
A Generic HPLC System




                        8
Generic HPLC Pump




                    9
    Controlling your HPLC Pump
•   Flow rate
•   Minimum and maximum pressure
•   Purge valves
•   Mobile phase mixtures




                                   10
Injector




           11
  Variable Wavelength &
Photodiode Array Detectors




                             12
                Column Heaters
Some separations of complex mixtures must be temperature
optimised to achieve separations of overlapping peaks.

Increased column temperature will also shorten retention times
for a given column dimension.

Selectivity of the column is also a function of temperature and
elution orders of peaks may change and even reverse - some
chiral and amino acid separations are very sensitive to column
temperature effects.

Eliminates retention time variation due to room temperature
fluctuations.                                               13
Column Heaters




                 14
Bits & Bobs




              15
Bits and Bobs




                16
            If Your Fittings Leak
•   Check to make sure your tubing is seated properly

• The fitting may not be tightened enough

• You may be using incompatible fittings

• Check the condition of the nut and ferrule

• Sometimes a leaking connection has nothing at all to do with the
  nut and ferrule, but with the receiving port

• NOTE: Using fittings made of material that is incompatible with
  your mobile phase is a sure way of creating leaks

                                                                    17
Bits & Bobs




              18
       Lesson 2

Running a simple separation




                              19
            “Normal Phase”
+ - + - + - + - + - + - + - + - + - + - + - +

     Mobile Phase -NON POLAR



+ - + - + - + - + - + - + - + - + - + - + - +

Stationary Phase - POLAR
                                          20
             “Reverse Phase”

      Mobile Phase - POLAR
                 +
         -                -
  +
                      +
                               -

Stationary Phase - NON POLAR
                                   21
           Reverse Phase


                                              Silica
                                              Stationery
                                              Phase
                                              support




The longer the alkyl chains, the longer the
retention time in a reversed phase column.          22
     Why Degas the Mobile Phase?


               Actual nitrogen
               concentration




                              Theoretical saturation

100% water-----------------------------------------------100% Methanol
                                                                  23
What is a Solvent Front?




                           24
    How to Degas the Mobile Phase
•   Sonicate.
•   Apply partial vacuum while agitating.
•   Helium sparge.
•   In-line degasser.




                                            25
               Gradient




Methanol -   Acetonitrile -   Ethyl acetate
ammonium     water
acetate
                                              20 minutes
                                                    26
             Things to try….
• Mobile phase composition. What happens to
  retention times and peak width?
• Pump speed. What happens to peak width?
• Column temperature. What happens to retention
  times?
• For Homework: Prepare a written report
  explaining your findings and include neatly
  tabulated data. Calculate column efficiency using
  the same analyte on each first run. You do not
  have to submit your chromatograms.
                                                  27
            Homework Report Format
                                                                 Column
            Retention   Peak width                  Peak width
Parameter                            Retention                   efficiency
            time.       at half                     at half
that was                             time.                       (analyte #1)
            (analyte    height                      height
changed                              (analyte #2)
            #1)         (analyte #1)                (analyte #2)




                                                                           28
       Peak Width at Half-height
• Peak width, or
  sharpness, is an
  indication of column
  efficiency.
• An ideal peak is a
  Gaussian distribution.
• The ratio of standard
  deviation to retention
  time is independent of
  flow rate.
                             tr    29
    Calculating Column Efficiency
• Peak width at half height is used to calculate
  column efficiency.

• N = 5.54 ( T / W )2

Where N = theoretical plates
W = Width at half height T = Retention time
5.54 is a constant based on the normal distribution

                                                      30
          Lesson 3

       Changing Columns
         Sample Loops
Care and Maintenance of Injectors



                                    31
Installing a Sample Loop




                           32
Routine Care of Injectors
             • Never use a pointed or
               bevel tip needle.
             • Rinse after the use of
               buffer solutions.
             • Avoid abrasive particles
               by filtering samples before
               injection.
             • Use burr-free tubing to
               avoid metals shavings
               from getting into the
               injector.

                                        33
Routine Care (continued)




                           34
    Leak Checking the Injector
• Leaks between stator   • Loosen set screws or
  and stator ring?         tighten stator screws.
• Leaks in needle port   • Tighten needle seal by
  ONLY when loading?       pushing in the guide.
• Fluid escapes by
  siphoning out?         • Adjust the height of
                           the outlet tubes.
• Continual leak out
  needle port or vent    • Surface of the rotor is
  lines?                   scratched, replace.

                                                 35
               Load Volume
• For a partial fill, use
  no more than 50% of
  the loop volume. The
  sample liquid tends to
  expand and fills the
  loop completely.
• To completely fill the
  sample loop, use at
  least 200% of the loop
  volume.
                             36
  Flushing the Needle Port Tube
• Use the manufacturer’s recommended
  adapter and connect it to a luer lock type
  syringe.
• Flush in the INJECT position only so that
  the fluid flows out the vent line.
• The sample loop will be flushed with
  mobile phase.

                                               37
How Do I Know My Injector is
         Clogged?




                               38
  Flushing the Stator and Stator
              Face
• With the pump turned off, disconnect the
  one sample loop fitting and the column
  connection.
• Using a syringe, gently pump a cleaning
  fluid (high purity methanol or water)
  through the ports.


                                             39
   Lesson 4
Care of HPLC Pumps




                     40
                    Pump Care
• Flush with water after running a buffer, (note
  there are special procedures when using reverse phase
  columns.)
• Replace seals in a timely manner.
• Maintain check valves.
• Do not allow solids in the mobile phase.




                                                          41
      Removing Buffers from a
       Reverse Phase Column
• DO NOT FLUSH WITH 100% WATER AS
  YOUR FIRST STEP -
• Substitute water for the buffer but leave the
  remaining proportions the same. Run through
  about 5 column volumes.
• Wash through 10 column volumes of a strong
  organic solvent, example - Methanol.
• If you plan to store the column, read the
  directions.
• If the phase collapses, a 50-50 water, organic
  solvent wash for 30 minutes can restore it.      42
  Lesson 5

HPLC Calibration




                   43
              Why Calibrate?
From an FDA Warning Letter of June 29, 2002
  issued to a Medical Gas supplier:
• 3. Failure to routinely calibrate mechanical and
  electronic equipment or keep records of calibration
  according to a written program designed to assure
  proper performance as required by 21 C.F.R. 211.68.
  For example, your electronic thermometer was last
  calibrated in June 1999, the pressure gauge for the
  manifold filler was last calibrated in February 1997,
  and the vacuum gauge has never been calibrated.


                                                      44
                            Why Calibrate?
    From an FDA Warning Letter of June 29, 2004 issued to a
    company that makes metals used in dental implants:
…not in conformity with the Current Good Manufacturing Practice (CGMP) requirements of the
Quality System (QS) regulation found at Title 21, Code of Federal Regulations (CFR), Part 820.
Significant violations include, but are not limited to, the following:

1. Failure to establish and maintain procedures to ensure that equipment is routinely calibrated,
inspected, checked, and maintained, as required by 21 CFR 820.72(a). For example, your written
procedures require that equipment be calibrated two times a year. The following equipment has not
been calibrated or checked two times a year: the…machine used to test the yield strength, ultimate
tensile strength, and elongation on the has not been calibrated. The next calibration date was
supposed to have been January 28, 2004.

2. Failure to document the equipment identification, calibration dates, the individual performing each
calibration, and the next calibration date, as required by 21 CFR 820.72(b)(2). For example, the
calibration data sheet dated August 26, 2003, for the spectrophotometer used to analyze other
metals was not signed and dated by the individual who performed the calibration and had no
specified test value for the “Std. Dev. Sample Units”.

                                                                                                  45
                    Calibration
•   wavelength accuracy,
•   wavelength precision (repeatability)
•   absorbance linearity
•   absolute absorbance accuracy.




                                           46
 Automated Detector Calibration

Filter with a known
Absorbance(s)

                      Flow cell


                              To waste

                       From column

                                         47
 Automated Detector Calibration

Filter with a known
Absorbance(s)

                      Flow cell


                              To waste

                       From column

                                         48
Holmium Oxide




                49
Agilent 1100 Wavelength
      Calibration

                486 nm
                             656.1 nm
1 nm slit



            Deuterium lamp
                                        50
            Manual Calibrations
                                 450 nm

                            410 nm



                                           640 nm

Linearity with a solution   Wavelength accuracy
of potassium dichromate,    with a solution of
0.01 to 0.20 mg/mL          Holmium oxide
                                                    51
     Compendial Requirements for
            Calibration.
• There must be written SOP’s.
• A calibration schedule must be maintained.
• All standards and meters must be NIST traceable.
  Any electrical meters, stopwatches, thermometers,
  also require calibration!
• Persons performing the calibration must be trained
  and this training must be documented.
• All equipment must be tagged with the date the
  calibration was performed, the person performing
  the calibration, and the date of the next calibration.
                                                      52
          Laboratory Exercise
• Remove the connection from the head of the
  column and cap the column end. Calibrate the
  flow of the pump at 1, 3, and 5 mL/min. using a
  stopwatch and graduated cylinder.
• Calibrate the injector by performing ten replicate
  injections of caffeine, calculate the average peak
  area and the relative standard deviations of all
  injections. Use enough standard to completely fill
  the sample loop.

                                                   53
       Homework for Calibration
•    Prepare a calibration report.
•    Include, the following information:
1.   The serial number, date calibrated, and date of next calibration
     for the stopwatch.
2.   The peak areas for the replicate caffeine injections, the average
     peak area, and the relative standard deviation for all ten
     injections.
3.   The manufacturer, lot number, and purity of the caffeine
     standard.
4.   Your report must be checked by another member of the class
     before it is submitted and that person must sign off on the report.

                                                                       54
      Lesson 6

Examples of HPLC in the
Pharmaceutical Industry



                          55
     Some Important Concepts
• What is a check standard?
• What is a system suitability standard?
• What is a five-point calibration?
• How does an internal standard work?
You will not be able to complete your HPLC
  lab reports unless you can answer these
  questions!
                                             56
“The most important assets of any
business are intangible: its company
name, brands, symbols, and slogans, and
their underlying associations, perceived
quality, name associations, customer
base and proprietary resources such as
patents, trademarks, and channel
relationships.”
          -David Aaker, author of several books on branding including:
                                       Managing Brand Equity, 1991




                                                                   57
     Courtesy Dr. Mark Kay, MSU Dept. of Business.
Intangible Assets as Percentage
           of Value.




                                                 58
 Courtesy Dr. Mark Kay, MSU Dept. of Business.
Intangible Assets and Brand




                                                59
Courtesy Dr. Mark Kay, MSU Dept. of Business.
Flu Vaccine and the Winter of
        2004 - 2005
          THE CHIRON PLAN
 1.   Chiron acquires British vaccine maker
      Powder Ject Pharmaceuticals.
 2.   Efficiency at the Liverpool plant is
      increased by 50%.
 3.   Sales of flu vaccine will finance an
      expansion into new vaccines and biotech
      drugs.

                                                60
     Flu Vaccine and the Winter of
             2004 - 2005
             THE CHIRON REALITY
1.    Chiron acquires British vaccine maker Powder Ject
      Pharmaceuticals.
2.    Efficiency at the Liverpool plant is increased by 50%.
3.    CHIRON FUMBLES QUALITY
4.    Citing contamination problems, British regulators
      suspend the plant’s license in October 2004.
5.    Without sales of flu vaccine Chiron loses $300 million
      in revenues. Earnings fall 50% from 2003. The stock
      price falls 30%.
                                                           61
    Chief Executive Officer Howard Pien said his company was
 surprised when British regulators suspended the Liverpool plant’s
                              license.

• Inspectors from the British
  Health Authority found
  contamination from
  “dangerous bacteria”
• FDA inspections revealed
  similar problems in 1999,
  2001, 2002, and 2003
• Chiron failed to correct
  the problems volentarily.


                                                                 62
Or as convicted counterfeiter
 Elsworth Roston once
 observed:


“Quality is your security.”

                                63
        Examples of HPLC in the
        Pharmaceutical Industry
• Manufacturing:           • Discovery:
• Content Uniformity       • Purity and concentration
• Degradation products     • Redox potential
  and related substances   • Structural confirmation
• Dissolution              • Development and
• Stability Studies          clinical trials
                           • Metabolite monitoring
                           • Half life in the body

                                                   64
Content Uniformity




                     65
                   Content Uniformity

Exampleamine HCl   Exampleamine HCl


Exampleamine HCl   Exampleamine HCl


Exampleamine HCl   Exampleamine HCl


Exampleamine HCl   Exampleamine HCl


Exampleamine HCl   Exampleamine HCl




                                        66
             Content Uniformity
• From an FDA Warning Letter of December
  2003 issued to a manufacturing facility:
• Regarding your Advicor product, your QCU failed to property
  investigate Content Uniformity failures found in lots of Advicor
  tablets. Specifically, lots of Advicor 500mg/20mg and lots of
  Advicor 750mg/20mg failed Content Uniformity release testing
  and were rejected. Your QCU attributed the failures to low "dew
  points". However, your "dew point" specification is no more than
  ___. The investigation into these failures did not evaluate lots
  released to the market that had been produced under similar
  conditions with similar dew points



                                                                 67
        Degradation and Related
             Compounds




• Degradation products may be formed from heat,
  bacterial action, or excessive moisture.
• Related compounds are known impurities created
  as artifacts of manufacturing.
                                                   68
Dissolution
  Exampleamine HCl




                     69
                          Dissolution
• From an FDA Warning Letter of December 2003
  issued to a manufacturing facility:
•   Your QCU attributed the Niaspan dissolution failures seen in lots of
    finished product to a low Hydroxypropyl content in the Methocel E10M
    raw material. Additionally, Niaspan lots produced using Methocel
    E10M lot #0207200002 also failed dissolution and these failures were
    also attributed to a low Hydroxypropyl content in the raw material. The
    Hydroxypropyl contents in both circumstances were within your NDA
    listed specification. This is of particular concern since the Methocel raw
    material helps to control the rate of release of the drug in your Niaspan
    product. It is also unclear if your QCU has assessed all of the
    manufacturing variables needed to optimize your production
    process…your Process Evaluation Summary also identified an increase
    in the operating temperature as a contributing factor to the dissolution
    failures.

                                                                            70
Dissolution




              71
       Automated Dissolution




HPLC with autosampler



              Fiber optic system

                                   72
               Stability Assays
• The FDA Defines a Stability Assay as a:
“Validated quantitative procedure that can detect
  changes with time in the pertinent properties of a
  drug substance and drug product. A stability-
  indicating assay accurately measures the active
  ingredients without interference from degradation
  products, process impurities, excipients, or other
  potential impurities.”
Federal Register, 65, August 2000.
                                                   73
                  Stability Studies



3 months 6months one year   three years    five years

• All pharmaceutical manufacturers are required to
  periodically test stored samples of their products
  (sometimes they are subjected to high
  temperatures and moist environments) in order to
  determine their stability over long periods of time.
                                                        74
 Levothyroxine sodium is a synthetic
hormone used to treat thyroid disorders.

       Hyperpotent dosage - cardiac pain
       palpitations, cardiac arrhythmias,
       Increased risk of osteoporosis

         IDEAL DOSAGE LEVEL

       Subpotent dosage - depression,
       lack of concentration, fatigue, weight gain,
       edema, loss of concentration, and constipation

                                                        75
        Levothyroxine sodium
1 962 - FDA requires NDA filings for all new drug products
1982 - One manufacturer removes two inert ingredients
 and changes the coating process. Potency increases by
22 to 30%.
1982 - FDA sets up a commission to develop “stability
indicating” HPLC methods.
1982 - 1994 - FDA receives 58 adverse drug experience reports.
1991 - The first of ten recalls involving 150 lots and more than
100,000,000 tablets. All recalls were due to subpotency or
because potency could not be guaranteed through the expiration
date.
                                                          76
        Levothyroxine sodium
1993 - FDA issues Warning Letter to manufacturer. Five lots of
tablets failed stability testing. Another lot was recalled because
potency could not be guaranteed through the expiration date.
1993 - Another manufacturer recalls six lots. Recalled tablets
tested 75 to 90% potency (USP requirements are 90 to 110%).
Another lot was recalled after failing routine stability testing.
1994 - Seven lots recalled because potency could not be assured
through the expiration date.
1995 - 60 lots and 50,436,000 tablets recalled
1995 - 22 lots recalled because potency could not be assured
through the expiration date.
                                                             77
        Levothyroxine sodium
The customary 2 year shelf life of most pharmaceuticals is
   inappropriate for Levothyroxine sodium because:

1. It is unstable in the presence of warm temperatures, light, air,
   and humidity. Some of these stability problems can be
   corrected through better control of the manufacturing
   environment.
2. Some of the excipients used in the tablets can catalyze
   degradation reactions.
3. The drug’s degradation occurs in two phases. An initial high
   degradation rate is followed by a slower degradation. To
   compensate, some manufacturers increase the tablet’s potency.
   This sometimes leads to hyperpotent doses.               78
    Example applications in Drug
            Discovery
• Compound purity and concentration prior to
  testing against a therapeutic target.
• Redox potential to estimate solubility and
  biological availability.
• Molecular weight confirmation



                                           79
Purity and Concentration




                           80
                   Redox Potential
• Electrochemical detectors are
  used when the analytes do not
  have good chromophores.

• This detector measures the
  current resulting from
  oxidation/reduction reaction of
  the analyte at a suitable
  electrode. Since the level of the
  current is directly proportional
  to the analyte concentration,
  this detector is used for
  quantification


                                      81
                   Redox Reactions

•   The pKa of a molecule is the pH at which half of the molecules
    (dissolved in water) are in one ionized form, with the other half being
    unionized or in a higher ionized form. pKa Therefore indicates the form
    a molecule will take at a given pH value, for example stomach or blood
    pH.

•   Some properties are pKa-dependent, such as lipophilicity, solubility and
    permeability.

•   pKa is important for understanding how a charged drug interacts with a
    receptor. When drugs metabolize they form ionized, hydrophilic
    metabolites which are excreted at physiological pH.



                                                                          82
         “Structural” Confirmations
• Many large compound
  libraries have been created
  for drug discovery programs.
• Robot assisted synthesis or
  Combinatorial Chemistry
  can create hundreds or even
  thousands of compounds in a
  short time.
• There is therefore a need to
  rapidly confirm the identity
  of these new compounds.



                                      83
      High Throughput HPLC
• Drug discovery costs are now topping
  2000$ per minute
• In a typical screen, as many as 250,000
  compounds are tested against a disease
  target
• Most drug discovery operations perform an
  HPLC analysis of every compound in their
  collection

                                              84
Example




          85
Example




          86
The BRIO System

       24 lanes with reversed
       phase packing.

       Eight channel
       autosampler for
       loading

       Reusable
                                87
 LC/MS Monitoring of Drugs in
          Blood
• Clinical trials for schizophrenia medications
  and other anti-psychotic drugs
• Half life in blood from 6 to 36 hours
• Agilent LC/MS systems with Zorbax C-18,
  30 mm X 4.6 mm columns. 3.5 micrometer
  stationary phase.


                                              88
 LC/MS Monitoring of Drugs in
          Blood
• Start with 0.5 mL serum
• Add internal standards
• Add organic solvents (hexane, n-butanol,
  acetonitrile,93:5:2)
• Evaporate
• Reconstitute in 50 uL MeOH (a ten-fold increase
  in concentration)
• Run in SIM mode (target masses between 313 and
  388, run times under four minutes.)
                                                89
The Future of Instrumentation and
        the FDA: 21CFR11
                or…
 Is a Paperless Laboratory Really
             Practical?




                                    90
                  21CFR11

 Beyond the buzzwords there is one simple concept:
                   SECURITY.
1. If users can save data, they can delete data
2. Centralized networks have centralized security
3. Central data stores can have different levels of
   access for different persons, chemists,
   supervisors, metrologists, and maintenance.

                                                      91
The Big Issues with 21CFR11
 1. Computer system security
 2. Backup and restoration
 3. Automated audit trails
    capturing the date, time, and
    user’s name any time that data
    is generated.
 4. Secure electronic signatures
                                     92
   “Simple” Steps to Implement
            21CFR11
• Be honest with people, explain to them at the start
  of the project that they will be monitored and set
  down clear rules about what will and will not be
  allowed with the new system.
• One mechanism from one vendor, not multiple
  systems from multiple vendors.
• No more individual access levels on individual
  computers.

                                                    93
   Software Mission Statements
• When starting any software project, write a one-
  page, easy to understand, simple, and concise
  explanation of what the software is supposed to
  do.
• Compare the real world performance of the system
  against the expectations set down in this
  document.
• Make this document available to the FDA should
  they ever want to audit or inspect your data
  systems.
                                                94
      Homework for Lesson 6
• Read a journal article that describes the use
  of HPLC in a pharmaceutical application. It
  can be from any sector of the industry but
  should describe a specific application in
  some detail.
• Write a one page summary of the article.


                                              95
     Lesson 7

Introduction to the USP




                          96
  Lesson 7
Retention factors
  Dead volume
   Resolution
 Tailing Factor
System suitability

                     97
            Retention Factor k

Retention                     retention time - dead time
              =      --------------------------------------------
Factor                        dead time


         Dead time = dead volume / flow rate

 (for a 4.6 mm ID column dead volume is approximately:
  0.1 X L where L is the column length in centimeters.

                                                                98
     Using the Retention Factor
• The retention factor is a measure of the how much
  the analyte is retained on the column compared to
  an unretained analyte.
• Ideally k should be between 2 and 20, and not less
  than 1.
• Values less than 1 usually indicate that the
  analysis is too sensitive to mobile phase organic
  composition, impurities, and injection effects.


                                                   99
   An LC/MS Example of the
 Importance of Retention Factors
• Problem: Suppression of early eluting
  compounds by the solvent front and the
  early eluting impurities.

                              K < 1, BAD




                                           100
  An LC/MS Example of the
Importance of Retention Factors




                  K < 2, GOOD


                                101
          Calculating Resolution
Selectivity = k (analyte 2) / k (analyte 1)

                    2 (RT B - RT A)
Resolution = ------------------------------------
              Peak width A + Peak Width B

               Baseline resolution has a value of 1.5 or
                higher.
                                                      102
             Tailing Factor (Tf)

• Tf = (peak width) / 2 (front’s half width)
• All widths measured at 5% peak height.
• Values greater than 1.5 generally indicate that
  unwanted interactions are occurring.




                                                    103
            Tailing Factor

                             Front width
                              Peak width




5% height


                                       104
         Asymmetry Factor (AS)

                             Back half width
                      AS = ----------------------
                             Front half width



10% height




                                                105
 Why System Suitability?
  “They are used to verify that the
  resolution and reproducibility of
chromatographic system are adequate
for the analysis to be done. The tests
 are based upon the concept that the
equipment, analytical operations, and
the samples to be analyzed constitute
    an integral system that can be
          evaluated as such.”
                                         106
         Why System Suitability?
• In November of 1998, the FDA issued this warning to a cosmetics /
  skin care products firm in California:

• 5. Your firm failed to establish and document the
  accuracy, sensitivity, specificity, and reproducibility of
  test methods used for raw materials, in process, and
  finished product testing. Specifically, your company has
  not validated laboratory methods used on various tests
  used in testing raw materials and sunscreen finished
  product. In addition, system suitability testing is not
  performed on the HPLC testing performed on the drug
  products. [CFR 211.165 (e)]

                                                                      107
      System Suitability is Taken
        Seriously by the FDA
  From an FDA Warning Letter sent to a pharmaceutical
  manufacturing company in New Jersey:

• 12. Written procedures containing provisions for
  remedial action in the event of laboratory
  instrumentation failing to meeting accuracy and
  precision limits are not followed (21 CFR
  211.160(b)(4)). For example, on three occasions,
  corrective actions following HPLC system suitability
  failures were not recorded in the instrument
  maintenance logs as required by your SOP.

                                                         108
              More System Suitability
• From a Warning Letter to a company the manufactures
  sunscreen and vitamins dated November 1998:

•   5. Your firm failed to establish and document the accuracy, sensitivity,
    specificity, and reproducibility of test methods used for raw materials, in
    process, and finished product testing. Specifically, your company has
    not validated laboratory methods used on various tests used in testing
    raw materials and sunscreen finished product. In addition, system
    suitability testing is not performed on the HPLC testing performed on
    the drug products. [CFR 211.165 (e)]




                                                                            109
      Lab Exercise for Lesson 7
• Run five injections of a two component mixture,
  each at a decreasing concentration. Example: 100
  mg/ml - 10 mg/ml - 1 mg/ml etc. etc.
• Measure the resolution, tailing factor, peak width
  at half-height.
• Report your results using the accepted standards
  of documentation.


                                                   110
         Lesson 8

Introduction to FDA Regulation




                                 111
  HPLC Problems Mentioned on
     FDA Warning Letters.
• No identification of mobile phase or solution.
• No identification of raw data and chromatograms.
• No identification of actual method used, especially
  when multiple methods are available.
• Procedures that did not test for degradation
  products or impurities.
• Failure to implement changes when HPLC
  methods have been found deficient.
• Failed system suitability with no records of the
  corrective action.
                                                   112
 Poor HPLC Methods, Example #1
• From a Warning Letter to Searle, December 1992:
• According to some records reviewed during the
  inspection, you developed a stability indicating method in
  1981 for Winstrol and in 1988 for Plaquenil capable of
  detecting degradation products. However, neither of the
  methods have been used for this even though you have
  data to show that degradation products and/or impurities
  can elute at long retention times. Both aforementioned
  methods are only used for assay determination with a
  limited run time of 9 minutes



                                                           113
       Changing a Bad Method.
• Changes are always tightly controlled to insure that
  data is valid from year to year and batch to batch.
• Proposed method changes are submitted in writing
  to a change control committee.
• The changes to the method are tested. Sometimes
  this is done in the same lab that uses the method
  every day, other companies have a separate group
  that performs this function.
• If approved, the new method is issued. Old copies
  of the method are collected and destroyed.
                                                     114
    Out of Specification (OOS)

Upper control limit


                       + 2 standard deviations
                               Mean value
                       - 2 standard deviations



 Lower control limit
                                          115
    Other Topics From the CFR
• Calibration procedures, records, and intervals.
• OOS Results (Testing into compliance).
• Uncontrolled changes to methods.
• Old procedures not collected and destroyed.
• Incomplete or absent review of data and
  records.
• No signatures on laboratory reports.


                                                116
Lab Exercises for Lesson 8

   Content Uniformity Assay




                              117
Lesson 9
Ion Pairing




              118
              Ion Pairing




In running HPLC analyses of ionized molecules,
 the mobile phase pH is often used to control the
amount of charge on the molecule,(and thus the
polarity). This is sometimes called “ion
suppression.”                                       119
          Ion Suppression
                       Moderate pH
     H+
H+                           H+
          H+
                 H+


     H+                     High pH

 Low pH

                  H+                  120
              Ion Pairing




In ion pairing, a molecule with a long alkyl chain
and terminal ionization sites is introduced into
the mobile phase.
                                                     121
              Ion Pairing


                                              Silica
                                              Stationery
                                              Phase
                                              support




The longer the alkyl chains, the longer the
retention time in a reversed phase column.          122
      Ion Pairing




Courtesy Regis Technologies 1998.
                                    123
     Lesson 10

Performing a Tablet Assay




                            124
 Lesson 11

Troubleshooting




                  125
                 Baseline Noise
• During the injection, there
  is a slight interruption in
  mobile phase flow. (A)
• Air bubbles in the system
  may cause irregular flow,
  in which case the size of
  the noise will be
  proportional to the flow
  rate. (B)
• If electronic noise in the
  detector is the cause, then
  the noise level will be
  independent of the flow
  rate. (B)                       126
     Example of an Air Bubble
            Problem
• Dr. Toney’s sample    • Using 01. ml/min on
  number 9746.            the LC/MS system
• Running a molecular   • A leaking fitting
  weight confirmation     failed, causing
                          pressure to drop and
                          allowing air into the
                          system.



                                                  127
      Example of an Air Bubble
             Problem

                        Fitting failure

Good, stable baseline
                             Air bubbles in system.




                                                      128
      Periodic baseline fluctuations, especially if
    accompanied by pressure fluctuations, may be
     caused by an air bubble in the pump or some
               other pump malfunction.
•  If the pump speed is     • Does the problem
   changed does the           persist even if there is
   fluctuation frequency      no flow?
   change?                  • Check detector
1. Prime the pump again     • Check for
                              environmental
2. Degas the mobile           influences such as
   phase                      power supplies, radios,
3. Perform routine pump       etc.
   maintenance              • Is the column
                              temperature uniform?
                                                     129
                Peak Fronting
• Overloaded column.
• Channels in the solid
  phase.




                                130
Peak Tailing
      • Silanol interactions
      • Degradation of stationary
        phase
      • Unswept void volume, or
        void formation at head of
        column,
      • Co-eluting material
      • POOR MATCH
        BETWEEN ANALYTE,
        MOBILE PHASE, AND
        COLUMN POLARITIES
                                131
       Overloading the Column
•   Loss of resolution   • Injection volume less
                           than 15% of peak
•   Tailing                volume.
•   Broadening           0.5 min X 0.3 ml/min = 0.15 ml
                         0.5 min X 3 ml/min = 15 ml
•   Retention time       • 1 uG of sample per cm
    decreases              of column length for a
                           4.6 mm ID column


                                                          132
             Matrix Overload
• In this situation,
  impurities in the
  sample matrix coat the
  column packing and
  reduce the resolution
  and the retention
  times.
• This can occur when
  samples are extracted
  from biological fluids.
                               133
                Ghost Peaks
• Column                • Reverse and flush
  Contamination           column
• Elution of analytes   • Use a longer run time,
  from a previous run     a different clean up, or
                          a strong solvent at the
                          end of the run



   First run                   Second run
                                                134
      Ghost Peaks from Ion Pairs
• Sometimes ghost         • This results in an ion
  peaks can appear from     pair formation that
  having a sample           causes the analyte to
  prepared in one           remain on the
                            stationary phase too
  solvent and using         long.
  another solvent as
                          • Ion pairing is a
  your mobile phase.        technique that
                            increases retention
                            time.
                                                     135
  Ghost Peaks or Random Baseline
              Noise?
• Sometimes seemingly random baseline
  noise can sometimes have the same cure as
  ghost peaks, turn the column around and
  flush it.




                                          136
Peak doubling or splitting

Sample volume too large Inject sample prepared in mobile phase with total
sample volume <15% of volume of first peak

Injection solvent too strong Use weaker injection solvent

Column void or channeling Replace column; use less-aggressive conditions

Blocked column frit Replace frit; add in-line filter; filter samples

Unswept injector flow path Replace injector rotor




                                                                       137
                 Drifting Baselines
       One run                            Days or weeks




• A suddenly change in the baseline (hours) during an
  isocratic separation usually indicates column or detector
  contamination.
• A slowly changing baseline (days or weeks) usually
  indicates a detector or a source problem.

                                                              138
Lesson 11 Continued

  More Troubleshooting




                         139
          High Backpressure
• All HPLC pumps have a pressure gauge
• If pressure rises it usually means a clogged
  frit or column
• The inlet frit on most columns is about 2
  microns.
• Reverse column and flush it.


                                             140
            Column Care Tips
Q: How do I now which solvents are safe to use on
  my column?
A: Read the directions
Q: How do I select a good solvent to flush my
  column?
A: Read the directions
Q: I want to store my column, what should I fill it
  with?
A: Read the directions
This may be a hard lesson for us guys to understand.141
         Air Bubbles Revisited
• Symptoms that might appear
Irregular baselines
Increasing run times
Low to moderate pressure drop
• Corrective actions
Degas mobile phase prior to a run and purge all air
   bubbles from lines.
Use an in-line degasser.
If problems persist, open purge valve and run
   system.                                          142
                    Pump Care
• Flush with water after running a buffer, (note
  there are special procedures when using reverse phase
  columns.)
• Replace seals in a timely manner.
• Maintain check valves.
• Do not allow solids in the mobile phase.




                                                          143
      Removing Buffers from a
       Reverse Phase Column
• DO NOT FLUSH WITH 100% WATER AS
  YOUR FIRST STEP -
• Substitute water for the buffer but leave the
  remaining proportions the same. Run through
  about 5 column volumes.
• Wash through 10 column volumes of a strong
  organic solvent, example - Methanol.
• If you plan to store the column, read the
  directions.
• If the phase collapses, a 50-50 water, organic
  solvent wash for 30 minutes can restore it.      144
        Drifting Retention Times
•   Flow rate changes (see pump care)
•   Improperly calibrated gradient controllers
•   Air bubbles
•   Temperature changes
•   Leaks
•   Column damage
•   Changes to mobile phase composition

                                                 145
      Drifting Retention Times
Changes to mobile phase composition

• Improper mixing either preparing the mixture
  in a batch or with a proportioning pump.
• Evaporation of a volatile component (very
  rare.)
• Incorrect pH


                                             146
 Strategy for Reducing HPLC Analysis Time
       and Maintaining High Resolution
                  Courtesy Agilent Technologies

1. Reduce column length.
2. Select a shorter column that has a smaller particle size.
(In 1969 particle sizes were typically 100 microns which gave 170
plates per 5 cm., by 2003 particle sizes were down to 1.8 microns
which gave 12,000 plates per 5 cm.)
3. Increase mobile phase temperature
(Increasing temperature reduces analysis time by as much as 40%
and pressure as much as 25%)
4. Increase flow rate.
                                                              147
Column Packing, Extracolumn
 Effects and Peak Resolution
                               LOSS of
                               resolution
                               due to
                               tubing
                               volume




Column packing particle size
                                            148
 Peak Broadening as a Result of
     Extracolumn Volume
• Made worse by
  short and narrow
  columns
• Smaller packings
  are more
  vulnerable


                                  149
      Homework for Lesson 11

• Read one Troubleshooting column from
  LC-GC Magazine.
• Write a summary of the article.
• Be prepared to present your summary to the
  class.



                                           150
Student Presentations




                        151
Semester Review




                  152
  What I have Done Wrong May
               ‘05
• Biggest mistake was not reviewing all 150 slides
  before doing the lesson plans, forgetting key
  points for resolution and column efficiency.
  Sometimes things planned for later in the class
  were needed sooner and I did not remember them
  correctly.
• Do not assume that, students know how an
  internal standard works, they can do a calibration
  curve, they can understand normal distribution.
• Explain system suitability and check standards
  slowly and carefully before the labs begin.       153

				
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