Advance Usp Methods

1U S P m e t h o d s

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3U S P m e t h o d s

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4 Introduction

APPLICATION INDEX

COMPOUND PAGE NO.

Acetaminophen 10,11Albuterol 14Alprazolam 12,13,71p-Aminobenzoic acid 65Amitriptyline HCl 15Amoxicillin 16, 72Aspirin 10, 73Atenolol 17, 18, 19Benzalkonium chloride 20Benzoic acid 10, 30, 38, 39Benzophenone 48Betamethasone 63, 64, 82Butyrophenone 56Caffeine 10Cefuroxime axetil 21Cephalexin 22, 23, 24, 74, 75Cephradine 24, 75Chloramphenicol 25, 76Cortisone acetate 26, 27Desipramine 50, 51Dextroamphetamine 61Dextromethorphan HBr 28Diphenhydramine 29Dopamine HCl 30Doxepin HCl 31Doxylamine succinate 32Epinephrine bitartrate 60Estradiol 33Estrone 33Ethinyl estradiol 34Ethyl paraben 33, 34, 57Fluoxetine HCl 35Glyburide 36, 37

COMPOUND PAGE NO.

Guaifenesin 38, 39Hydrocodone bitartrate 40Hydrocortisone 41, 42, 77Hydrocortisone acetate 43, 78Hydrocortisone cream 44, 451-Hydroxybenzatriazole 22, 23, 74Ibuprofen 46, 47, 48, 79Imipenem (sterile) 49Imipramine 50, 51Lidocaine HCl 52, 80Lorazepam 53Medroxyprogesterone 54, 55Methyl paraben 52, 80Minoxidil 54, 55Naproxen 56, 57Nortriptyline 58, 59Oxacillin 81Phenylephrine HCl 60Phenylpropanolamine 61Prednisolone 63, 64, 82Prednisone 41, 42, 62, 77Procainamide HCl 65, 67Progesterone 36, 37Propoxyphene 66Propranolol 67Pseudoephedrine HCl 29Reserpine 68Salicylic acid 10, 73Tetracaine 69Thiamphenicol 83Triazolam 12, 13, 71Valerophenone 46, 47, 79

5Introduction

TABLE OF CONTENTS

Application Index ----------------------------------------------- 4

About this guide ------------------------------------------------- 6I. Method Validation --------------------------------------- 7II. Adjusting USP Methods -------------------------------- 8

USP Methods ---------------------------------------------------- 9

Alternative Methods -------------------------------------------- 70

Notes --------------------------------------------------------------- 84

U S P m e t h o d s

6Our goal in developing this guide was to illustratesome of the ways which USP/NF methods may be"modified" in order to improve system performance orincrease sample throughput. The requirements ofmany of the validated HPLC methods have been leftintentionally vague in order to allow some room formodification to fit specific circumstances. For instance,a method may state that the flow rate should be"around 1 mL/min". This vagueness would imply thatmodifications to the flow rate are acceptable, so longas the modified method still meets system suitabilityrequirements. This can be a great advantage toanalysts who, for whatever reason, are unable to meetsystem suitability requirements under the specifiedmethod. For instance, closely eluting peaks which failsystem suitability resolution requirements may beresolved through slight modifications to mobile phasepH, or even by going to a longer column or smallerparticle size packing for a higher efficiency separation.In addition, simple modifications such as increasingflow rate or reducing column length can significantlyincrease sample throughput over the long run.

ABOUT THIS GUIDE

However, this vagueness has also led to considerableconfusion as to exactly what is considered "modified"versus "changed". In response to this, several articleshave been published, and the USP itself has pub-lished a proposed list of acceptable system modifica-tions in Pharmacopeial Forum vol. 25(2) and vol. 26(5).This list, once approved, will be added to the SystemSuitability section of the USP.

Although we cannot perform your validations for you,we can act as a resource for columns and informationto better utilize USP methods. With this guide and ourLuna HPLC columns you will be able to comply withUSP requirements, as well as modernize and optimizeyour methods for improved performance and in-creased productivity.

Introduction

7Chromatographic methods are routinely employed in labsaround the world for a variety of analytical and preparatorypurposes. Due to the relative ease with which most samplescan be analyzed and the robustness of many chromato-graphic analyses, these techniques have become themethods of choice for combinatorial screening of potentialdrug products to routine QA/QC analysis of nutritionalproducts.

In order to ensure the reproducibility and ruggedness ofdeveloped methods, a process known as method validationis carried out. Validation, as defined by the USP is "theprocess of providing documented evidence that the methoddoes what it is intended to do" and is performed on allinstruments and methods used in an analytical method.Thus, everything from the software and hardware system tothe method itself must be validated before the analyticalmethod is considered "validated". However, the focus of thisguide is specifically on the validation of HPLC methods asdefined in the USP/NF.

Method validation for HPLC methods consists of a processdesigned to provide documentation that the method willperform as intended. This process of 8 steps or "AnalyticalPerformance Parameters" (USP {1225}) which must bedetermined in order for a method to be considered "vali-dated". The parameters to be determined are:

1. The accuracy of a method refers to "the closeness of testresults obtained by that method to the true value" (USPXXIII). In most cases an accuracy value of 98-102%,determined by calculating percent recovery values of aknown standard or against a second technique, is required.In most pharmaceutical analyses, accuracy is determinedagainst a standard curve generated using known amountsof target analyte.

2. Precision represents the degree of variability in yourresults using the same method. This is normally expressedas relative standard deviation (RSD%) and must be lessthan 2% for most methods. There are three different levelsof precision:

a. Method precision (repeatability) is determined bymultiple analyses of the same sample over a shortperiod of time.

I. METHOD VALIDATION

b. Intermediate precision is the precision of the methodwhen run on different days by different operators usingdifferent instruments.

c. Reproducibility is the variability when the samemethod is performed by different labs.

3. Specificity is the "ability to measure accurately andspecifically the analyte in the presence of components thatmay be expected to be present in the sample matrix" (USPXXIII). In other words, the ability to separate your targetcompound from interfering components. There can be nointerference from known impurities or degradation/stressproducts.

4. The linearity or range of a method refers to the ability ofa method to give accurate results over a given range ofanalyte concentrations. Generally, your assay must have acorrelation coefficient >0.997 for 5 points over 50-150% ofyour expected target analyte concentration.

5. Limit of detection is a value given to the lowest amountof analyte that can be detected, but not quantified, using agiven method. Often, this minimum value is specified asthree times the signal-to-noise ratio.

6. Limit of quantitation, as the name implies, representsthe lowest concentration of analyte which can be accuratelyquantified using a given method. This value is usually aminimum of 10 times the signal-to-noise ratio.

7. The ruggedness of a method is "the degree of repro-ducibility of the test results obtained by the analysis of thesame sample under a variety of normal test conditions,such as different laboratories, different analysts, differentinstruments, different lots of reagents, different elapsedassay times, different assay temperatures, different days,etc." (USP XXIII). In other words, will other labs be able toreproduce your results? Typically, a valid method must havean RSD% of less than 2% between labs.

8. Method robustness is a measure of the ability of amethod to withstand small changes in running conditions.You demonstrate method robustness by varying factorssuch as mobile phase pH, flow rate, temperature, etc.

Introduction

8 Why should I make modifications to USP methods?Although the methods written in the USP compendia haveall gone through the validation process and so should, inthe best of circumstances, represent methods which willreliably and reproducibly produce acceptable results for themethods, there are instances where it may be worthinvestigating changing or modifying existing methods. Forinstance, it could be that a particular formulation for a drugsubstance gives co-elution with an excipient under the USPconditions. A method which was validated using oneparticular formulation cannot take into account all of thepossible excipients which may be present in future formula-tions. Thus, it may be possible that a given USP methodsimply will not resolve the target analyte with acceptablespecificity due to excipient compounds present in aformulation.

Productivity issues / saving time may also be validconsiderations for USP methods. Many of these methodswere written using very old HPLC technology, and thus maydepend on the use of long columns (300 mm) with largeparticle sizes (10). Given the numerous advances incolumn technology, many of these older methods could bemore efficiently performed using modern HPLC columnswith smaller, more highly efficient (3) particles and shortercolumn lengths. Over the long run, the time savingsresulting from using shorter columns can be substantial.

Improved results can also be an added benefit of usingmore modern, state-of-the-art HPLC column technologies.The vast advances in silica quality and bonding techniqueshave resulted in columns which, for many applications, willperform substantially better than their older predecessors.For example, columns packed with low-purity silicas maydisplay extensive tailing with basic or acidic compounds,and failure of the column to meet system suitability require-ments for a given assay may delay production and productrelease.

As stated previously, many USP HPLC methods are leftintentionally vague. This may be advantageous in that itallows some flexibility in a method, allowing the analyst to"fine-tune" and adjust it to certain situations. If you do makeadjustments which are within a reasonable range, you donot have to entirely revalidate, although you must show animprovement in chromatography using reference standards.In addition, it may be advisable to show equivalence to anexisting, validated method. The problem arises in determin-ing just what an "acceptable" modification to a methodconsists of, and when a modification becomes a "change"

II. ADJUSTING USP METHODS

which would warrant re-validation of a method. For instance,if a method calls for a 300 x 4.6mm column, do I have to re-validate if I choose to use a 250 x 4.6mm? What about a150 x 4.6mm column?

In response to these types of questions, several adjustmentlimits have been proposed and reviewed in recent publica-tions. At this point, minor modifications to USP methods arecommonly used and accepted so long as they conform tothe method in intent. The following acceptable method"modifications" have been proposed (Pharmocopeial Forum25(2) and 26(5)) and are as follows :

1. Mobile phase pH: 0.2 units pH of 7.6 can be adjusted from 7.4-7.8

2. Concentration of salts in buffer: 10% 20mM Potassium phosphate can be 18-22mM, as

long as proper pH is maintained as above.

3. Ratio of components in mobile phase: 30%of the minor component(s), or 2% absolute of thatcomponent, whichever is greater. However a change inany component cannot exceed 10% absolute, norcan the final concentration be reduced to zero.

60:40 Acetonitrile/Water can be adjusted to 12%water (=30% of 40), but this exceeds the 10%maximum absolute change, so can range from 30%to 50% water in this case

4. Wavelength of UV-Visible Detector: no deviationspermitted

5. Column length: 70% 150 x 4.6 mm column can be varied 105 mm in

length

6. Column inner diameter: 50% 150 x 4.6 mm column can be varied 2.3 mm

7. Particle size: can be reduced as much as 50% 10 can be switched with a 5 particle

8. Flow rate: 50% 1 mL/min can be varied from 0.5 to 1.5 mL/min

9. Injection volume: increased to as much as twice thevolume specified, provided no adverse effects

Must be within stated linearity range of method!

10. Column temperature: 20C

Introduction

9USP METHODS

USP Methods

10

Standard prep: Benzoic acid 0.36mg/mL, all others at 0.1mg/mLMobile phase: Water / Methanol / Glacial acetic acid (69:28:3)

Flow rate: 2.0 mL/minDetection: UV @ 275nm

Temperature: 45CInjection: 10 L

Sample: 1. Acetaminophen2. Caffeine3. Aspirin4. Benzoic acid5. Salicylic acid

0021

System Suitability RequirementUSP Tailing Factor for all < 1.2Resolution between any analyte and I.S. is > 1.4

USP column specified: 100 x 4.6mm L1Column used: 100 x 4.6mm 5m C18(2)Part No.: 00D-4252-E0

USP/NF 23 page:

USP Methods

- Acetaminophen, aspirin and caffeine - USP Method 2A00710

APP. ID No 3270

Also See ALTERNATIVE METHOD page 73

11

USP/NF 23 page:

Standard prep: Acetaminophen - 0.01mg/mL in mobile phaseMobile phase: Water / Methanol (75:25)

Flow rate: 1.5 mL/minDetection: UV @ 243nmInjection: 10 L

0017

System Suitability RequirementEfficiency(N) > 1000 platesUSP Tailing Factor < 2.0

Column Performance:Efficiency(N) = 5047 plates/columnUSP Tailing Factor = 1.14Resolution = NA

USP column specified: 300 x 3.9mm L1Column used: 250 x 4.6mm 5m C18(2)Part No.: 00G-4252-E0

USP Methods

2A00200 - Acetaminophen capsules - USP Method

APP. ID No 3259

12

USP/NF 23 page:

USP Methods

APP. ID No 3262

Standard prep: Alprazolam and Triazolameach 0.025mg/mL in mobile phase

Mobile phase: Acetonitrile / Chloroform / Butanol / Water / Acetic acid(850 : 80 : 50 : 20 : 0.5)


Sample: 1. Triazolam2. Alprazolam

- Alprazolam - USP Method 2A03940

System Suitability RequirementResolution > 2.0

Column Performance:Efficiency(N) = 8940USP Tailing Factor = 1.14Resolution = 7.05

USP column specified: 300 x 4.6mm L3Column used: 250 x 4.6mm 5m SilicaPart N0.:00G-4042-E0

0046

13

USP/NF 23 page:

USP Methods

APP. ID No 3276

Standard prep: Alprazolam and Triazolameach 0.025mg/mL in mobile phase

Mobile phase: Acetonitrile / Chloroform / Butanol / Water / Acetic acid(850 : 80 : 50 : 20 : 0.5)



- Alprazolam - USP Acceptable Modified Method

NA



USP column specified: 300 x 4.6mm L3Column used: 100 x 4.6mm 5m Silica(2)Part No.:00D-4274-E0


14

USP/NF 23 page:

USP Methods

APP. ID No 3260

- Albuterol Tablets - USP Method 2A02962

0039

USP column specified: 150 x 4.6mm L1Column used: 150 x 4.6mm 5m C18(2)Part No.:00F-4252-E0

Standard prep: Albuterol - 0.03mg/mL in mobile phaseMobile phase: Methanol / Water with 5mM Hexane sulfonic

acid and 1% Glacial acetic acid (40 : 60)Flow rate: 1.5 mL/minDetection: UV @ 276nmInjection: 20 L

System Suitability RequirementUSP Tailing Factor < 2.5Efficiency(N) > 800 (was 5647)

Column Performance:Efficiency(N) = 5647 Plates/columnUSP Tailing Factor = 1.10Resolution = NA

15

USP/NF 23 page:

USP Methods

APP. ID No 3319

Standard prep: Amitriptyline 0.2 mg/mL in waterMobile phase: Acetonitrile / 92 mM Monobasic

sodium phosphate pH 2.5 (42:58)Flow rate: 2 mL/minDetection: UV @ 254 nmInjection: 20L

2A09600 - Amitriptyline Hydrochloride - USP Method

0093

System Suitability RequirementUSP Tailing Factor < 2.0 (was 1.56)Efficiency(N) > 800

Column Performance:Efficiency(N) = 4812USP Tailing Factor = 1.56Resolution = NA

USP column specified: 300 x 3.9mm L1Column used: 300 x 3.9mm Bondclone 10m C18Part No.:00H-2117-C0

16

USP/NF 23 page:

- Amoxicillin - USP Method 2A11300

0100

USP column specified: 250 x 4.6mm L1Column used: 250 x 4.6mm 5m C18(2)Part No.:00G-4252-E0

Standard prep: Amoxicillin - 1.2mg/mL in phosphate bufferMobile phase: 50mM Potassium phosphate pH 5.0 /

Acetonitrile (96 : 4)Flow rate: 1.5 mL/minDetection: UV @ 230nmInjection: 10 L

System Suitability RequirementUSP Tailing Factor < 2.5 Efficiency(N) > 1700k between 1.1-2.8


USP Methods

APP. ID No 3258

17

Standard prep: Atenolol 0.2mg/mL in citrate buffer pH 6.0Mobile phase: Acetonitrile / 5mM 1-Octanesulfonatic acid

with 38mM Sulfuric acid (25 : 75)Flow rate: 1.7 mL/minDetection: UV @ 275 nmInjection: 10 L

2A17776 - Atenolol Injection - USP Method

NA

System Suitability RequirementUSP Tailing Factor < 2.0


USP column specified: 250 x 4.6mm 5m L1Column used: 250 x 4.6mm 5m C18(2)Part No.:00G-4252-E0

USP/NF 23 page:

USP Methods

APP. ID No 3284

18

USP/NF 23 page:

Standard prep: Atenolol 0.01mg/mL in mobile phaseMobile phase: Methanol / 5mM Dibasic sodium phosphate

with 7mM 1-Heptanesulfonate and 20mMDibutylamine, pH 3.0 (300 : 700)


- Atenolol - USP Method 2A17774

NA

System Suitability RequirementUSP Tailing Factor < 2.0Efficiency(N) > 5000



USP Methods

APP. ID No 3285

19

- Atenolol - USP Acceptable Modified Method

0320


Standard prep: Atenolol 0.01mg/mL in mobile phaseMobile phase: Methanol / 5mM Dibasic sodium phosphate

with 7mM 1-Heptanesulfonate and 20mMDibutylamine, pH 3.0 (300 : 700)

Flow rate: 0.9 mL/min (was 0.6mL/min in USP)Detection: UV @ 226nmInjection: 10 L



USP/NF 23 page:

APP. ID No 3286

USP Methods

20

USP/NF 23 page:

USP Methods

APP. ID No 9300

- Benzalkonium Chloride - USP Acceptable Modified Method 7B00500

2218

USP column specified: L10Column used: 150 x 4.6mm 5m CNPart No.:00F-4255-E0

Standard prep: Benzalkonium chloride - 4 mg/mL in waterMobile phase: 100mM Sodium acetate pH 5.0 / Acetonitrile

(40 : 60)Flow rate: 2 mL/minDetection: UV @ 254nmInjection: 1 L (was 20L in USP)

Sample: 1. C12 peak2. C14 peak

System Suitability RequirementEfficiency(N) > 1000 platesResolution > 1.5

Column Performance:Efficiency(N) = 7552 plates/columnUSP Tailing Factor = 1.61 and 1.27Resolution = 2.70

21

USP/NF 23 page:

APP. ID No 3318

USP Methods

Standard prep: Cefuroxime axetil 0.24mg/mL in mobile phaseand acetanilide 0.54mg/mL in mobile phase

Mobile phase: Methanol / 0.2M Monobasic Ammonium Phosphate (380 : 620)Flow rate: 1.0 mL/min (was 1.5mL/min in USP)Detection: UV @ 278nmInjection: 10 L

Sample: 1. Acetanilide2&3. Cefuroxime axetil diastereomers

2C08820 - Cefuroxime Axetil - USP Acceptable Modified Method

0315


Column Performance:Efficiency(N) = 8442 plates/columnUSP Tailing Factor = 0.93Resolution = 2.69

USP column specified: 250 x 4.6mm 5m L13Column used: 250 x 4.6mm Develosil TMS-UG 5mPart No.: CH0-4230

22

USP/NF 23 page:

USP Methods

APP. ID No 3280

Standard prep: Cephalexin (0.2 mg/mL) and 1-hydroxybenzotriazole (0.1mg/mL)in mobile phase

Mobile phase: Water / Acetonitrile / Methanol / Triethylamine (850 : 100 : 50 : 15)with 5mM 1-Pentanesulfonic acid and adjusted to pH 3.0


Sample: 1. 1-Hydroxybenzotriazole2. Cephalexin

- Cephalexin - USP Method 2C09200

0320





23

USP/NF 23 page:

APP. ID No 3279

USP Methods

Standard prep: Cephalexin (0.2mg/mL) and 1-hydroxybenzotriazole(0.1 mg/mL)in mobile phase

Mobile phase: Water / Acetonitrile / Methanol /Triethylamine (850 : 100 : 50 : 15)with 5mM 1-Pentanesulfonic acid and adjusted to pH 3.0



- Cephalexin - USP Acceptable Modified Method

0320




24

USP/NF 23 page:

USP Methods

APP. ID No 3264

Standard prep: Mix of cephradine and cephalexin,each 0.1mg/mL in mobile phase

Mobile phase: Water / Methanol / 0.5 M Sodium acetate /0.7 N Acetic acid (782 : 200 : 15 : 3)


Sample: 1. Cephradine2. Cephalexin

- Cephradine capsules - USP Acceptable Modified Method 2C10100

0326

System Suitability RequirementResolution cephalexin and cephradine must be > 2.0


USP column specified:250 x 4.6mm 10m L1Column used: 250 x 4.6mm 5m C18(2)Part No.:00G-4252-E0


25

USP/NF 23 page:

APP. ID No 3312

USP Methods

2C11500 - Chloramphenicol - USP Acceptable Modified Method

0332

System Suitability RequirementUSP Tailing Factor > 2.0


USP column specified: 100 x 4.6mm 5m L1Column used: 100 x 4.6mm 5m C18(2)Part No.:00D-4252-E0

Standard prep: Chloramphenicol at 80 g/mL in mobile phaseMobile phase: Water / Methanol / Glacial acetic acid

(55 : 45 : 0.1)Flow rate: 1.0 mL/minDetection: UV @ 280nmInjection: 10 L


26

USP/NF 23 page:

Standard prep: Cortisone acetate - 0.1 mg/mLMobile phase: Water / Acetonitrile (55 : 45)

Flow rate: 2 mL/minDetection: UV @ 254nmInjection: 35 L

- Cortisone acetate - USP Method 2C27200

0428

System Suitability RequirementEfficiency(N) > 1500k > 2.0



USP Methods

APP. ID No 8709

27

USP/NF 23 page:

- Cortisone acetate - USP Acceptable Modified Method

0428

System Suitability RequirementEfficiency(N) > 1500k> 2.0


USP column specified: 300 x 3.9mm 5m L1Column used: 150 x 4.6mm 5m C18(2)Part No.:00F-4252-E0

Standard prep: Cortisone acetate 0.1 mg/mLMobile phase: Water / Acetonitrile (55 : 45)

Flow rate: 2.0 mL/minDetection: UV @ 254nmInjection: 20 L (was 35L in USP)

APP. ID No 8710

USP Methods

28

USP/NF 23 page:

USP Methods

APP. ID No 8711

Standard prep: Dextromethorphan HBr-0.1 mg/mLin mobile phase

Mobile phase: Acetonitrile / Water (70 : 30), both with7mM Docusate sodium and 7mMAmmonium nitrate, pH 3.4 with acetic acid


- Dextromethorphan HBr - USP Method 2D06700

0482


Column Performance:Efficiency(N) = 22328USP Tailing Factor = 1.08Resolution = NA


29

USP/NF 23 page:

APP. ID No 11203

Standard prep: Diphenhydramine and Pseudoephedrine@ 25ug/mL in 0.5% acetic acid

Mobile phase: Methanol / Acetonitrile / Water w/10mM Heptanesulfonate and 13mM Triethylamine,pH 3.3 (10 : 26 : 64)


Sample: 1. Pseudoephedrine2. Diphenhydramine

2D17030 - Diphenhydramine & Pseudoephedrine - USP Method

0534

System Suitability RequirementResolution between two > 3.0USP Tailing Factor < 2.0

Column Performance:Efficiency(N) = 9460 plates/column for diphenhydramine,

8867 for pseudoephedrineUSP Tailing Factor = 1.16 for diphenhydramine,

0.96 for pseudoephedrineResolution = 21.96

USP column specified: 250 x 4.6mm L10Column used: 250 x 4.6mm 5m CNPart No.:00G-4255-E0

USP Methods

30

USP/NF 23 page:

USP Methods

APP. ID No 8712

- Dopamine HCl Injection - USP Acceptable Modified Method 2D0400

0549




Standard prep: Dopamine 0.16mg/mL and benzoic acid 0.5mg/mL in mobile phaseMobile phase: Water with 1% Acetic and 5mM

Octanesulfonic acid / Acetonitrile (87 : 13)Flow rate: 2.0 mL/min (was 1.5 by USP)Detection: UV @ 280nmInjection: 40 L (was 351 in USP)

Sample: 1. Benzoic acid2. Dopamine

31

USP/NF 23 page:

APP. ID No 9230

USP Methods

Standard prep: Doxepin 100g/mL in mobile phaseMobile phase: 0.2m Monobasic sodium phosphate / Methanol (61 : 39),

adjusted to pH2.5 with phosphoric acid (USP method was 70 : 30)Flow rate: 1 mL/minDetection: UV @ 254nmInjection: 20 L

Temperature: 50CSample: E and Z isomers of Doxepin

2D20800 - Doxepin HCl - USP Method

0551

System Suitability RequirementResolution > 1.5USP Tailing Factor < 2.0

Column Performance:Efficiency(N) = 14342 plates/columnUSP Tailing Factor = 1.16 and 1.31 for isomersResolution = 2.24

USP column specified: 125 x 4.6mm L7Column used: 125 x 4.6mm 5m C8(2)Part No.:00E-4249-E0

32

USP/NF 23 page:

USP Methods

APP. ID No 3274

Standard prep: Doxylamine 0.25mg/mL in mobile phaseMobile phase: Acetonitrile / Water (37 : 63) with 25mM

Monobasic Potassium phosphate, 10mMTriethylamine, and 5mM Sodium lauryl sulphate


- Doxylamine succinate - USP Acceptable Modified Method 2D22300

0560



USP column specified: 150 x 4.6mm L7Column used: 100 x 4.6mm 3m C8(2)Part No.: 00D-4248-E0

33

USP/NF 23 page:

APP. ID No 8713

USP Methods

2E07900 - Estradiol - USP Acceptable Modified Method

0622

System Suitability RequirementResolution between Estradiol and estrone > 2.0

Column Performance:Efficiency(N) = 22223 plates/column for estradiolUSP Tailing Factor = 1.15 for estradiolResolution = 11.52 for 3/2, and 8.88 for 2/1


Standard prep: Ethyl paraben-0.75mg/mLEstradiol-20g/mLEstrone-33g/mL

Mobile phase: Acetonitrile / Water (55 : 45)Flow rate: 1.0 mL/min (was 1.5 by USP)Detection: UV @ 205nmInjection: 25 L

Sample: 1. Ethyl paraben2. Estradiol3. Estrone

34

USP/NF 23 page:

USP Methods

APP. ID No 3315

Standard prep: Ethinyl estradiol-200g/mLEthyl paraben-20g/mL

Mobile phase: Water / Acetonitrile (50 : 50)Flow rate: 1.0 mL/minDetection: UV @ 280nmInjection: 25 L

Sample: 1. Ethinyl estradiol2. Ethyl paraben

0638


Column Performance:Efficiency(N) = 10128 plates/column for ethinylestradiolUSP Tailing Factor = 1.32Resolution = 13.30


- Ethinyl estradiol - USP Method 2E10800

35

USP/NF 23 page:

APP. ID No 3273

USP Methods

Standard prep: Fluoxetine HCl-110g/mL in mobile phaseMobile phase: 10mM Triethylamine buffer pH 6.0 w/phosphoric acid /

Tetrahydrofuran / Methanol (60 : 30 : 10)Flow rate: 1.0 mL/minDetection: UV @ 227nmInjection: 10 L

2F04840 - Fluoxetine HCl - USP Method

Supplement No. 8p. 4210




36

USP/NF 23 page:

USP Methods

APP. ID No 3316

Standard prep: Glyburide (0.5mg/mL) and progesterone(0.2mg/mL) in mobile phase

Mobile phase: Acetonitrile / 10 mM Monobasic ammoniumphosphate, pH 5.25 (55 : 45)


Sample: 1. Glyburide2. Progesterone

- Glyburide - USP Method 2G2930

0713


Column Performance:Efficiency(N) = 9853 plates/columnUSP Tailing Factor = 1.0 for glyburideResolution = 6.57


37

USP/NF 23 page:

APP. ID No 3317

USP Methods

Standard prep: Glyburide (0.5mg/mL) and progesterone(0.2mg/mL) in mobile phase

Mobile phase: Acetonitrile / Water with 10mM Monobasicammonium phosphate, pH 5.25(550 : 450)

Flow rate: 1.0 mL/min (was 2.0mL/min in USP)Detection: UV @ 254nmInjection: 10 L

Sample: 1. Glyburide2. Progesterone

- Glyburide - USP Acceptable Modified Method

0713


Column Performance:Efficiency(N) = 15428 plates/column for glyburideUSP Tailing Factor = 0.94Resolution = 8.08

USP column specified: 250 x 4.6mm L7Column used: 125 x 4.6mm 3m C8(2)Part No.:00E-4248-E0

38

USP/NF 23 page:

USP Methods

APP. ID No 3281

Standard prep: Guaifenesin-40g/mLBenzoic acid-100g/mL

Mobile phase: Water / Methanol / Acetic acid (60 : 40 : 1.5)Flow rate: 2.0 mL/minDetection: UV @ 276nmInjection: 20 L

Sample: 1. Guaifenesin2. Benzoic acid

- Guaifenesin Tablets - USP Method 2G05800

0724


Column Performance:Efficiency(N) = 3734 plates/column for guaifenesinUSP Tailing Factor = 1.04Resolution = 10.83


39

USP/NF 23 page:

APP. ID No 3267

USP Methods

Standard prep: Guaifenesin-40g/mL Benzoic acid-100g/mLMobile phase: Water / Methanol / Acetic acid (60 : 40 :1.5)


Sample: 1. Guaifenesin2. Benzoic acid

- Guaifenesin Tablets - USP Acceptable Modified Method

0724


Column Performance:Efficiency(N) = 3870 plates/column for guaifenesinUSP Tailing Factor = 1.23 for guaifenesinResolution = 10.83

USP column specified: 250 x 4.6mm 10m L1Column used: 100 x 4.6mm 5m C18(2)Part No.:00D-4252-E0

40 USP Methods

USP/NF 23 page:

APP. ID No 9299

Standard prep: Hydrocodone 1mg/mL in methanolMobile phase: Acetonitrile / Water / Diethylamine / Methanol

(440 : 2.2 : 0.55 : 45)Flow rate: 1.5 mL/minDetection: UV @ 280nmInjection: 5 L(USP was 20L)

- Hydrocodone bitartrate - USP Acceptable Modified Method 2H06000

0751

System Suitability Requirement


USP column specified: 250 x 4.6mm L3Column used: 250 x 4.6mm 5m Silica(2)Part No.:00G-4274-E0

41

USP/NF 23 page:

APP. ID No 9295

USP Methods

Standard prep: Hydrocortisone 0.1 mg/mL and prednisone 0.06 mg/mL in chloroformMobile phase: Butyl chloride / Water-saturated butyl chloride / THF / Methanol /

Glacial acetic acid (95 : 95 : 14 : 7 : 6)Flow rate: 1.5 mL/minDetection: UV @ 254nmInjection: 10 L

Sample: 1. Hydrocortisone2. Prednisone

2H06200 - Hydrocortisone - USP Method

0753




42 USP Methods

USP/NF 23 page:

APP. ID No 3287

Standard prep: Hydrocortisone 0.1 mg/mL and prednisone 0.06 mg/mL in chloroformMobile phase: Butyl chloride / Water-saturated butyl chloride / THF / Methanol /

Glacial acetic acid (95 : 95 : 14 : 7 : 6)Flow rate: 1.5 mL/minDetection: UV @ 254nmInjection: 10 L


- Hydrocortisone - USP Acceptable Modified Method 2H06200

0753




43

USP/NF 23 page:

APP. ID No 9298

USP Methods

Standard prep: Hydrocortisone acetate 0.1 mg/mLin chloroform

Mobile phase: Butyl chloride / Water-saturatedbutyl chloride / Tetrahydrofuran / Methanol /Glacial acetic acid (95:95:14:7:6)


2H07000 - Hydrocortisone acetate - USP Method

0758

System Suitability RequirementUSP Tailing Factor > 2.0


USP column specified: 300 x 4.0mm 10m L3Column used: 250 x 4.6mm 10m Silica(2)Part No.:00G-4091-E0


44

USP/NF 23 page:

USP Methods

APP. ID No 3271

Standard prep: Hydrocortisone-50g/mL in methanolMobile phase: Water / Acetronitrile (75 : 25)


- Hydrocortisone cream - USP Method 2H06300

0754

System Suitability RequirementNone stated

Column Performance:Efficiency(N) =13,569USP Tailing Factor =1.02Resolution = NA


45

USP/NF 23 page:

APP. ID No 8714

USP Methods

Standard prep: Hydrocortisone-50g/mL in methanolMobile phase: Water / Acetronitrile (75 : 25)

Flow rate: 1.5 mL/min (was 2mL/min USP)Detection: UV @ 254nmInjection: 15 L

- Hydrocortisone cream - USP Acceptable Modified Method

0754

System Suitability RequirementNone started


USP column specified: 300 x 3.9mm L1Column used: 100 x 4.6mm 3m C18(2)Part No.:00D-4251-E0


46

USP/NF 23 page:

USP Methods

APP. ID No 3278

Standard prep: Ibuprofen 12mg/mL and valerophenone 0.35mg/mL in mobile phaseMobile phase: Acetonitrile / Water (600:400) with 20mM Chloroacetic acid, pH3.0


Sample: 1. Ibuprofen2. Valerophenone

- Ibuprofen - USP Method 2I00100

0785




47

USP/NF 23 page:

APP. ID No 3277

USP Methods

Standard prep: Ibuprofen 12mg/mL andvalerophenone 0.35mg/mL in mobile phaseMobile phase: Acetonitrile / Water (600:400) with 20mM Chloroacetic acid, pH3.0



2I00100 - Ibuprofen - USP Acceptable Modified Method

0786





48

USP/NF 23 page:

USP Methods

APP. ID No 3263

Standard prep: Ibuprofen - 0.4 mg/mLBenzophenone - 0.3 mg/mL in mobile phase

Mobile phase: 0.01M phosphoric acid / Acetonitrile (55 : 45)(USP method states 63 : 37)


Sample: 1. Benzophenone2. Ibuprofen

- Ibuprofen Oral Suspension - USP Acceptable Modified Method 2I00150

0551

System Suitability RequirementResolution > 1.5Peak tailing < 2.0 (was 1.03)



49

USP/NF 23 page:

APP. ID No 3272

USP Methods

Standard prep: Imipenem 0.4 mg/mL in mobile phaseMobile phase: 7mM potassium phosphate pH 6.8


2I00770 - Sterile Imipenem - USP Method

0792

System Suitability RequirementEfficiency(N) > 600


USP column specified: 300 x 4.0mm L1Column used: 150 x 4.6mm AQUA 5m C18Part No.:00F-4299-E0

50

USP/NF 23 page:

USP Methods

APP. ID No 3255

Standard prep: Imipramine and desipramine 0.3mg/mLin mobile phase

Mobile phase: 0.06M Sodium perchlorate / Acetonitrile /Triethlamine (625 : 375 : 1), adjusted withPerchloric acid to a pH of 2.0


Sample: 1. Imipramine2. Desipramine

- Imipramine - USP Method 2I00800

0794




51

USP/NF 23 page:

APP. ID No 3248

USP Methods

- Imipramine - USP Acceptable Modified Method

0794




Standard prep: Imipramine and desipramine 0.3mg/mLin mobile phase

Mobile phase: 0.06M Sodium perchlorate / Acetonitrile /Triethlamine (625 : 375 :1),adjusted with Perchloric acid to a pH of 2.0

Flow rate: 2 mL/min (was 1.5 in USP)Detection: UV @ 269nmInjection: 20 L

Sample: 1. Imipramine2. Desipramine

52

USP/NF 23 page:

USP Methods

APP. ID No 3254

Standard prep: Lidocaine-1.7mg/mL in mobile phaseMethyl paraben-220g/mL in mobile phase

Mobile phase: Acetonitrile / Water with 5% Acetic acid, pH 3.4 (20:80)Flow rate: 1.5 mL/minDetection: UV @ 254nmInjection: 5 L (was 20 L in USP method)

Sample: 1. Lidocaine2. Methyl paraben

0887

System Suitability RequirementResolution Lidocaine/methyl paraben > 3.0

Column Performance:

Resolution = 22.57


- Lidocaine HCl - USP Acceptable Modified Method 2L02900Also See ALTERNATIVE METHOD page 80

53

USP/NF 23 page:

APP. ID No 3253

USP Methods

min1.0 2.03.0 4.0 5.0

2L05856 - Lorazepam Tablets - USP Method

0905




Standard prep: Lorazepam 0.1mg/mL in mobile phaseMobile phase: Water / Methanol / Acetic acid (55 : 45 : 2)


54

USP/NF 23 page:

USP Methods

APP. ID No 3266

Standard prep: Minoxidil 0.2 mg/mL and Medroxyprogesterone acetate 0.25 mg/mL in mobile phase

Mobile phase: Methanol / Water / Acetic acid (700 : 300 : 10) with7mM Docusate sodium and pH 3.0 with perchloric acid

Flow rate: 1.0 mL/minDetection: UV@254nmInjection: 10 L

Sample: 1. Minoxidil2. Medroxyprogesterone acetate

- Minoxidil - USP Method 2M23430

1032




55

USP/NF 23 page:

APP. ID No 3265

USP Methods

- Minoxidil - USP Acceptable Modified Method

1032




Standard prep: Minoxidil 0.2 mg/mL and Medroxyprogesterone acetate 0.25 mg/mLin mobile phase

Mobile phase: Methanol / Water / Acetic acid (700 : 300 : 10) with3.0g docusate sodium pH 3.0 with perchloric acid

Flow rate: 1.5 mL/min (was 1 mL/min in USP)Detection: UV @ 254nmInjection: 10 L

Sample: 1. Minoxidil2. Medroxyprogesterone acetate

56

USP/NF 23 page:

USP Methods

APP. ID No 3282

- Naproxen Tablets - USP Method 2N01900

1054

System Suitability RequirementEfficiency(N) > 4000Resolution > 11.0



Standard prep: Naproxen 25 g/mL and butyrophenone0.001L/mL

Mobile phase: Acetonitrile / Water / Glacial Acetic acid(50 : 49 : 1)


Sample: 1. Naproxen2. Butyrophenone

57

USP/NF 23 page:

APP. ID No 3283

USP Methods

2N01950 - Naproxen Oral Suspension - USP Acceptable Modified Method

1053

System Suitability RequirementResolution > 3.0USP Tailing Factor < 2.0

Column Performance:Efficiency(N) = 5175 plates/columnUSP Tailing Factor = 1.15 for naproxenResolution = 8.91


Standard prep: Naproxen - 50 g/mLEthylparaben - 4.4 g/mL

Mobile phase: Methanol / Water (50 : 50) with 30mM Sodium acetate pH 5.8Flow rate: 1.5 mL/minDetection: UV @ 254nmInjection: 20 L

Sample: 1. Naproxen2. Ethylparaben

58

USP/NF 23 page:

USP Methods

APP. ID No 9296

- Nortriptyline HCl Capsules - USP Method 2N12400

1107

System Suitability RequirementEfficiency(N) > 500USP Tailing Factor < 3.0



Standard prep: Nortriptyline 0.38mg/mL in methanolMobile phase: Acetonitrile / Methanol / 12mM Potassium

phosphate pH 6.7 (40 : 43 : 17)Flow rate: 2.5 mL/minDetection: UV @ 239nmInjection: 5 L

59

USP/NF 23 page:

APP. ID No 9297

USP Methods

- Nortriptyline HCl Capsules - USP Acceptable Modified Method

1107



USP column specified: 250 x 4.6mm L10Column used: 100 x 4.6mm 3m CNPart No.:00D-4254-E0

Standard prep: 0.38mg/mL in methanolMobile phase: Acetonitrile / Methanol / 12mM Potassium

phosphate pH 6.7 (40 : 43 : 17)Flow rate: 2.5 mL/minDetection: UV @ 239nmInjection: 5 L

60

USP/NF 23 page:

USP Methods

APP. ID No 3246

- Phenylephrine Hydrochloride Injection - USP Method 2P13200

1212


Column Performance:Efficiency(N) = 13430 plates/columnUSP Tailing Factor = 1.32 for epipnephrine, 0.78 for phenylephrineResolution = 7.32


Standard prep: Phenylephrine HCl and Epinephrine bitartrate,both 0.4mg/mL in mobile phase

Mobile phase: Water / Methanol (50 : 50) with 0.1%1-octanesulfonic acid adjust to pH3.0with phosphoric acid

Flow rate: 1.0 mL/minDetection: UV @ 280nmInjection: 5 L (USP was 20L)

Sample: 1. Epinephrine bitartrate2. Phenylephrine HCl

61

USP/NF 23 page:

APP. ID No 8275

USP Methods

1214




Standard prep: Phenylpropanolamine HCl and Dextroamphetamine sulfate, each5g/mL in mobile phase

Mobile phase: THF / Methanol / Water with 0.2% TMAH and 0.5% Phosphoric acid(4 : 40 : 956)

Flow rate: 1.5 mL/min (USP was 1.0mL/min)Detection: UV @ 215nmInjection: 10 L (was 5 L in USP)

Sample: 1. Phenylpropanolamine2. Dextroamphetamine

- Phenylpropanolamine HCl-Limit of Amphetamine- USP Acceptable Modified Method

62

USP/NF 23 page:

USP Methods

APP. ID No 3268

- Prednisone oral solution - USP Acceptable Modified Method 2N12400

1285

System Suitability RequirementUSP Tailing Factor< 2.0



Standard prep: Prednisone at 40g/mL in methanol / water(25 : 75)

Mobile phase: 17mM Monobasic potassium phosphate /Acetonitrile (60 : 40)


63

USP/NF 23 page:

APP. ID No 11202

USP Methods

2P23800 - Prednisolone - USP Method

1277




Standard prep: Presnisolone and Betamethasone, 0.1mg/mLin chloroform

Mobile phase: Butyl chloride / Water-saturated butyl chloride /Tetrahydrofuran / Methanol / Glacial acetic acid(95 : 95 : 14 : 7 : 6)


Sample: 1. Betamethasone2. Presnisolone

64

USP/NF 23 page:

USP Methods

APP. ID No 3269

- Prednisolone - USP Acceptable Modified Method

1230




Standard prep: Presnisolone and betamethasone, 0.1mg/mLin chloroform

Mobile phase: Butyl chloride / Water-saturated butyl chloride /Tetrahydrofuran / Methanol / Glacial acetic acid(95 : 95 : 14 : 7 : 6)


Sample: 1. Betamethasone2. Presnisolone

65

USP/NF 23 page:

APP. ID No 3261

USP Methods

2P28300 - Procainamide HCl - USP Method

1294




Standard prep: Procainamide HCl - 0.05mg/mL in mobile phasep-Aminobenzoic acid - 0.01mg/mL in mobile

phaseMobile phase: Water / Methanol / TEA (140 : 60 : 1) adjusted to

pH 7.5 with phosphoric acidFlow rate: 1.0 mL/minDetection: UV @ 280nmInjection: 20 L

Sample: 1. p-Aminobenzoic acid2. Procainamide HCl


66

USP/NF 23 page:

USP Methods

APP. ID No 3269

- Propoxyphene HCl Capsules - USP Acceptable Modified Method 2P30900

1319



USP column specified: 33 x 4.6mm 3m L1Column used: 50 x 4.6mm 3m C18(2)Part No.:00B-4251-E0

Standard prep: Propoxyphene 6.5g/mL in mobile phaseMobile phase: 50mM Monobasic potassium phosphate pH 3.0

with 20mM Triethylamine / Acetonitrile(70 : 30, was 3 : 2 in USP)

Flow rate: 1.0 mL/minDetection: UV @ 220nmInjection: 5 L (10L in USP)

Sample: 1. Propoxyphene

67

USP/NF 23 page:

APP. ID No 3291

USP Methods

2P31700 - Propranolol - USP Method

1327

System Suitability RequirementResolution > 2.0USP Tailing Factor for propranolol < 3.0



Standard prep: Propranolol (0.04mg/mL) and Procainamide(0.04mg/mL) in mobile phase

Mobile phase: Water / Acetonitrile / Methanol (70 : 70 : 90)with 7mM Sodium Lauryl sulfateand 11mM Phosphoric acid


Sample: 1. Procainamide2. Propranolol

68

USP/NF 23 page:

- Reserpine - USP Method 2R01000

1369




Standard prep: Reserpine at 10g/mL in mobile phaseMobile phase: Acetonitrile / 1% Ammonium chloride,

pH5.6 (50 : 50)Flow rate: 1.5 mL/minDetection: UV @ 268nmInjection: 20 L

USP Methods

APP. ID No 3292

69

USP/NF 23 page:

2T05300 - Tetracaine HCl opthalmic solution - USP Method

1506




Standard prep: 0.1mg/mL in waterMobile phase: 10mM Ammonium phosphate pH3.0 / Acetonitrile

(70 : 30)Flow rate: 2.0 mL/minDetection: UV @ 280nmInjection: 5 L

APP. ID No 3293

USP Methods

70

ALTERNATIVE METHODS

USP Alternative Methods

The following Alternative Methods are basedon USP methods but have been changed tooptimize runtime, peak shape and/or providemore rugged mobile phase conditions.

Because the methods are "changed" and not"modified" within the proposed guidelines ofPharmacopeial Forum vol. 25(2) and vol. 26(2),the following methods are not USP acceptedmodified methods.

These methods warrant additional validation bythe USP.

71

Standard prep: Alprazolam and Triazolam each 0.025mg/mLin mobile phase

Mobile phase: Hexane / Methylene choride / Methanol (75 : 20 : 5)Flow rate: 1.0 mL/minDetection: UV @ 254nmInjection: 20 L


USP column specified: 300 x 4.6mm L3Column used: 100 x 4.6mm 3m CNPart No.:00D-4254-E0

- Alprazolam - Alternative Method



APP. ID No 3309


72 USP Alternative Methods

- Amoxicillin - Alternative Method

System Suitability Requirementk between 1.1 -2.8Efficiency(N) > 1700USP Tailing Factor < 2.5



Standard prep: Amoxicillin - 1.2mg/mL in phosphate bufferMobile phase: 20mM Ammonium acetate pH4.9 / Methanol


APP. ID No 3294

73

APP. ID No 3301


- Aspirin tablets - Alternative Method




Standard prep: Aspirin 2mg/mL and Salicylic acid 1.6mg/mLMobile phase: 20mM Ammonium formate pH3.0 / Acetonitrile


Sample: 1. Aspirin2. Salicylic acid


APP. ID No 3308

- Cephalexin - Alternative Method



USP column specified: 250 x 4.6mm L1Column used: 150 x 4.6mm 5m Phenyl-HexylPart No.:00F-4257-E0

Standard prep: Cephalexin(0.2mg/mL) and 1-Hydroxybenzotriazole(0.1mg/mL) in mobile phase

Mobile phase: Water with 0.05%formic acid / Methanol with 0.05% formic acid (70 : 30)Flow rate: 1.5 mL/minDetection: UV @ 254nmInjection: 5 L


75

APP. ID No 3295


- Cephradine capsules - Alternative Method

System Suitability RequirementResolutionResolution > 2.0


USP column specified: 250 x 4.6mm 10m L1Column used: 150 x 4.6mm 5m C8(2)Part No.:00F-4249-E0

Standard prep: Mix of Cephradine and Cephalexin, each 0.1mg/mL in mobile phaseMobile phase: 20mM Ammonium acetete pH4.9 / Methanol (70 : 30)


Sample: 1. Cephalexin2. Cephradine

1


APP. ID No 3312

- Chloramphenicol - Alternative Method



USP column specified: 100 x 4.6mm 5m L1Column used: 50 x 4.6mm 3m C18(2)Part No.:00B-4251-E0

Standard prep: Chloramphenicol at 80g/mL in mobile phaseMobile phase: Water with 0.05%formic acid / Methanol with

0.05% formic acid (70 : 30)Flow rate: 1.5 mL/minDetection: UV @ 280nmInjection: 4 L

77

APP. ID No 3303


- Hydrocortisone - Alternative Method



USP column specified:Column used: 150 x 4.6mm 5m Phenyl-HexylPart No.:00F-4257-E0

Standard prep: Hydrocortisone 0.1mg/mL in mobile phase andPrednisone 0.06mg/mL in mobile phase

Mobile phase: Acetonitrile / Water (20 : 80)Flow rate: 2.0 mL/minDetection: UV @ 254nmInjection: 5 L



APP. ID No 3311

- Hydrocortisone Acetate - Alternative Method



USP column specified:Column used: 150 x 4.6mm 5m C8(2)Part No.:00F-4249-E0

Standard prep: Hydrocortisone acetate 0.1mg/mL in mobile phaseMobile phase: Methanol / Water (60 : 40)


79

APP. ID No 3278


- Ibuprofen - Alternative Method




Standard prep: Ibuprofen-12mg/mL in mobile phaseValerophenone-0.35mg/mL in mobile phase

Mobile phase: 20mM Ammonium acetate pH4.0 / Acetonitrile(60 : 40)




APP. ID No 3300

- Lidocaine HCl - Alternative Method

System Suitability RequirementResolution Lidocaine/methyl paraben > 3.0

Column Performance:Efficiency(N) = 4136 plates/column for LidocaineUSP Tailing Factor = 1.25Resolution = 11.16

USP column specified:Column used: 150 x 4.6mm 5m C8(2)Part No.:00F-4249-E0

Standard prep: Lidocaine-1.7mg/mL in mobile phaseMethyl paraben-220g/mL in mobile phase

Mobile phase: 20mM Ammonium acetate with 30mM TFA /Acetonitrile / Methanol (70 : 10 : 20)


Sample: 1. Lidocaine2. Methyl paraben

81

APP. ID No 3296


- Oxacillin - Alternative Method



USP column specified:Column used: 150 x 4.6mm 5m Phenyl-HexylPart No.:00F-4257-E0

Standard prep: Oxacillin 0.5mg/mL in mobile phaseMobile phase: Water / Acetonitrile / Methanol,

all with 10mM Formic acid (40 : 30 : 30)Flow rate: 1.5 mL/minDetection: UV @ 230nmInjection: 2 L


APP. ID No 3302

- Prednisolone - Alternative Method



USP column specified:Column used: 100 x 4.6mm 3m CyanoPart No.:00D-4254-E0

Standard prep: Prednisolone and Betamethasone,0.1mg/mL in chloroform

Mobile phase: Hexane / Isopropanol (85 : 15)Flow rate: 1.5 mL/minDetection: UV @ 254nmInjection: 10 L

Sample: 1. Betamethasone2. Prednisolone

83

APP. ID No 3297


- Thiamphenicol - Alternative Method



USP column specified:Column used: 50 x 4.6mm 3m C18(2)Part No.:00B-4251-E0

Standard prep: Thiamphenicol at 0.5mg/mL in mobile phaseMobile phase: Water with 0.05% formic acid / Methanol with

0.05% formic acid (85 : 15)Flow rate: 1.5 mL/minDetection: UV @ 280nmInjection: 20 L

84 Notes

85Notes

88

U S P m e t h o d s

USA411 Madrid Ave.Torrance, CA90501-1430USA(310) 212-0555(310) [email protected]

mail:

tel.:fax:

email:

Puerto Rico271 Sierra Morena,Suite #104San Juan,Puerto Rico 00926(800) 541-HPLC(310) [email protected]

Canada411 Madrid Ave.Torrance, CA90501-1430USA(800) 543-3681(310) [email protected]

United KingdomQueens Avenue,Hurdsfield Ind. Est.,Macclesfield, Cheshire SK10 2BN, [email protected]

GermanyZeppelinstr. 563741 AschaffenburgDeutschland

[email protected]

New ZealandP.O. Box 31-601MilfordAucklandNew [email protected]

AustraliaP.O. Box 264Pennant HillsNSW [email protected]

www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department by telephone, fax or e-mail: [email protected]. SM

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