HPLCSystem HA

Column: Silica Spherisorb S5W (125 × 4.9 mm i. d., 5 μm). Mobile phase: Solution containing 1.175 g (0.01 M) of ammonium perchlorate in 1 L

methanol; adjust to pH 6.7 by the addition of 1 mL 0.1 M sodium hydroxide in methanol. k values: Values for drugs in this system will be found in drug monographs and in the Indexes

to Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow.

System HX Column: Lichrospher 60 RP-Select B (125 × 4.0 mm i.d., 5 μm) with pre-column Lichrospher

60 RP-Select B (4 × 4.0 mm i.d., 5 μm). Mobile phase: (A:B) triethylammonium phosphate buffer (25 mM, pH 3.0):acetonitrile. Elution programme: (A:B) (100:0) to (30:70) in 30 min, hold 10 min, back to initial conditions

in 3 min with equilibration for 10 min before next injection. Flow rate: 1 mL/min. Detection: UV diode-array. Standards: Nitro-n-alkanes (C1 to C11) 10 μL in 10 mL acetonitrile. RI values: Values for drugs in this system will be found in the monographs and in the Indexes

to Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow.

System HY Column: C18 symmetry (250 × 4.6 mm i.d., 5 μm). Column temperature: 40°. Mobile phase: (A:B) sulfuric acid (0.5 mL of 2.5 M) in water (500 mL):sulfuric acid (0.5 mL

of 2.5 M) in acetonitrile (500 mL). Elution programme: (98:2) for 3 min to (2:98) over 23 min, hold for 10 min back to initial

conditions over 2 min with equilibration of 8 min before next injection. Detection: UV diode-array. Standards: Nitro-n-alkanes (C1 to C16) 10 μL in 10 mL acetonitrile. RI values: Values for drugs in this system will be found in the monographs and in the Indexes

to Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow.

System HZ Column: C18 endcapped LiChrospher 100 RP-18e, (125 × 4.0 mm i.d., 5 μm) with pre-column

LiChrocart 124-4. Mobile phase: Add 146 μL triethylamine and about 750 μL phosphoric acid to 530 mL water.

Adjust pH to 3.3 using a 10% potassium hydroxide solution & finally add 470 mL acetonitrile. Flow rate: 0.6 mL/min. Detection: UV diode-array. Retention times: Values for drugs in this system will be found in the monographs and in the

Indexes to Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow.

System HAA Column: C8 Symmetry (250 × 4.6 mm i.d., 5 μm) with Symmetry C18 pre-column (20 mm). Column temperature: 30°. Mobile phase: (A:B) phosphate buffer (pH 3.8):acetonitrile. Elution programme: (85:15) for 6.5 min to (65:35) until 25 min to (20:80) for 3 min and back

to initial conditions for equilibration for 7 min. Flow rate: 1 mL/min for 6.5 min, then linear increase to 1.5 mL/min for 6.5 to 25 min and hold

for 3 min (re-equilibration is made at 1.5 mL/min). Detection: UV diode-array. Retention times: Values for drugs in this system will be found in the monographs and in the

Indexes to Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow

System HBK Column: Lichrospher RP-8ec (250 × 4.0 i.d., 5 μm). Mobile phase: Three different composition are used: A: acetonitrile:phosphate buffer pH 2.3

(33:67). Internal standard: 5-(4-methylphenyl)-5-phenylhydantoin (for compounds eluting within 30 min); B: acetonitrile:phosphate buffer pH 2.3 (67:33). Internal standard: 4-phenylbenzophenone (for compounds eluting after 30 min); C: acetonitrile:phosphate buffer pH 2.3 (20:80). Internal standard: salicylamide (for compounds with RRTs below 0.2).

Flow rate: 1 mL/min. Detection: UV diode-array. Note: The phosphate buffer is prepared by dissolving 4.8 g phosphoric acid (85%) and 6.66 g

potassium dihydrogen phosphate in 1 L of water, adjust pH to 2.3. Values for drugs in this system will only be found in the Indexes to Analytical Data in Volume 2.

System HAD Column: C18 Symmetry Shield (250 × 4.6 mm i.d., 5 μm) protected by 2 μm Upchurch filter. Column temperature: 30°. Mobile phase: (A:B) M/15 potassium dihydrogen phosphate with 1% (v/v) octane sulfonic

acid:acetonitrile. Mobile phase (MP) 1: (95:5) at flow rate 1 mL/min; MP 2: (80:20) at flow rate 1 mL/min; MP 3: (30:70) at flow rate 1.2 mL/min.

Eluent switching programme: At injection, MP1 to the column. From time 12 to 30 min, MP2 to the column. From time 30 min, MP3 to the column to rinse it. From time 35 to 40 min, equilibration with MP1.

Detection: UV diode-array (λ=260 nm).

System HAF Column: ODS TSK-gel Super (100 × 4.6 mm i.d., 2 μm). Mobile phase: (A:B) Acetonitrile:sodium dihydrogen phosphate (5 mM, pH 6). Isocratic elution: (45:55). Flow rate: 0.65 mL/min. Detection: UV (λ=254 nm).

System HAV Column: RP-short alkyl chain, silanol deactivated (SCD 100) (250 × 4.6 mm i.d.), stainless

steel. Mobile phase: (A:B) Methanol:dibasic potassium phosphate (0.04 M, pH 5.5). Isocratic elution: (50:50). Flow rate: 1 mL/min. Detection: UV (λ=237 nm).

System HBA Column: C18 base-deactivated silica (125 × 4.6 mm i.d., 5 μm) with base-deactivated C18 pre-

column (20 × 4.6 mm i.d., 5 μm). Eluent: (A:B) Acetonitrile:potassium dihydrogen phosphate (50 mM, pH 7.5, containing

500 μL triethylamine). Isocratic elution: (60:40). Flow rate: 2 mL/min. Detection: Fluorescence (λex=255 nm, λem=315 nm).

System HBB Column: C18 (250 × 6.0 mm i.d., 5 μm). Eluent: (A:B) Acetonitrile:phosphate buffer (50 mM, pH 7.2). Isocratic elution: (43:57). Flow rate: 1.7 mL/min. Detection: Electrochemical (working electrode: glassy carbon, reference electrode: Ag/AgCl).

System HAE Column: C18 (Lichrospher, 100 RP-18, 5 μm) with C18 pre-column (Lichrospher RP-18, 5 μm). Mobile phase: (A:B) acetonitrile:sodium phosphate (25 mM) modified with diethylamine

(0.9%) and tetrahydrofuran (1%), pH 3.0. Isocratic elution: (44.8:55.2). Flow rate: 0.5 mL/min. Detection: UV (λ=260 nm).

System HAK Column: C18 Symmetry (250 × 4.6 mm i.d., 5 μm) with C18 pre-column Symmetry sentry. Mobile phase: (A:B) Acetonitrile:potassium dihydrogen phosphate (20 mM). Elution programme: (50:50) to (70:30) in 15 min. Flow rate: 1 mL/min. Detection: UV (λ=313 nm).

System HAL Column: C18 Novapak (150 × 4.6 mm i.d., 5 μm). Mobile phase: (A:B:C) Acetonitrile:methanol:phosphate buffer (6 mM), pH 5.7. Isocratic elution: (30:10:60). Flow rate: 1.3 mL/min. Detection: UV diode-array (λ=242 nm). Note: The phosphate buffer stock solution is prepared using 94 mL 0.2 M sodium dihydrogen

phosphate added to 6 mL 0.2 M disodium phosphate heptahydrate.

System HAM Column: C18 (150 × 4.0 mm i.d., 3 μm) with C18 pre-column (40 × 4.0 mm i.d., 3 μm). Mobile phase: (A:B) water:acetonitrile. Isocratic elution: (50:50). Flow rate: 0.7 mL/min. Detection: UV (λ=313 nm).

TLC system

No Mobile phase Chamber type

Stationary phase

Reference compounds* hRc

F system

(1) Chloroform–acetone (4:1) Saturated Silica gel

ParacetamolClonazepamSecobarbital

Methylphenobarbital

15355570

TD

(2) Ethyl acetate Saturated Silica gel

SulfathiazolePhenacetin

SalicylamideSecobarbital

20385568

TF

(3) Chloroform–methanol (9:1) Saturated Silica gel

HydrochlorothiazideSulfafurazolePhenacetinPrazepam

11335272

TAD

(4a)Ethyl acetate–methanol–25%

ammonia (17:2:1)Saturated Silica gel

Sulfadimidine Hydrochlorothiazide

Temazepam Prazepam

13 34 63 81 TE

(4b)Ethyl acetate–methanol–25%

ammonia (17:2:1)Saturated Silica gel

Morphine Codeine Hydroxyzine Trimipramine

20 35 53 80 TE

(5) Methanol Unsaturated Silica gel

CodeineTrimipramineHydroxyzine

Diazepam

20365682

TAE

(6)

Methanol–n-butanol (3:2) containing

0.1 mol/L sodium bromide

Unsaturated Silica gelCodeine

Diphenhydramine Quinine Diazepam

22 48 65 85 TAF

(7)Methanol–25%

ammonia (100:1.5)

Saturated

Silica gel impregnated

with 0.1 mol/L KOH in

methanol and dried

Atropine Codeine Chlorprothixene

Diazepam

18 33 56 75 TA

(8)

Cyclohexane– toluene–

diethylamine (15:3:2)

Saturated

Silica gel impregnated

with 0.1 mol/L KOH in

methanol and dried

Codeine Desipramine Prazepam

Trimipramine

6 20 36 62 TB

(9) Chloroform–methanol (9:1)

Saturated Silica gel impregnated

with 0.1 mol/L KOH in

Desipramine Physostigmine Trimipramine

Lidocaine

11 36 54 71

TC

TLC system

No Mobile phase Chamber type

Stationary phase

Reference compounds* hRc

F system

methanol and dried

(10) Acetone Saturated

Silica gel impregnated

with 0.1 mol/L KOH in

methanol and dried

Amitriptyline Procaine Papaverine Cinnarizine

15 30 47 65 TL

GAS CROMATOGRAFI

System GA Packed column: 3% SE-30 or OV-1 on 80 to 100 mesh Chromosorb G HP (acid washed and

dimethyldichlorosilane treated), 2 m × 2 mm i.d. glass column; it is essential that the support be fully deactivated.

Column temperature: normally between 100° and 300°; for isothermal conditions, an approximate guide to temperature is to use the RI ÷ 10.

Carrier gas: nitrogen at 45 mL/min. Capillary column: 10 to 15 m × 0.32 or 0.53 mm i.d., 100%-dimethyl-PSX (X-1) with a 1.5 to

3 μm film thickness. Carrier gas: helium. Temperature programme: 4 min at 135°, 13°/min to 200°, 6°/min to 312°, 6 min final hold.

System GB Capillary column: 20 to 30 m × 0.2 or 0.25 mm i.d., 5%-phenyl–95%-dimethyl-PSX (X-5) with

a 0.5 to 1 μm film thickness. Carrier gas: helium, constant flow 1 mL/min. Temperature programme: 0.7 min at 90°, 35°/min to 240°, 8°/min to 290°, 25°/min to 325°,

6 min final hold. Reference compounds: n–alkanes with an even number of carbon atoms, or a reference drug

mix that contains amfetamine (1125), ephedrine (1365), benzocaine (1545), methylphenidate (1725), diphenhydramine (1870), tripelennamine (1976), methaqualone (2135), trimipramine (2215), codeine (2375), nordazepam (2490), prazepam (2648), papaverine (2825), haloperidol (2930) and strychnine (3116). (RIs for system GA are given in parentheses for the drug mix.)

Retention indices: values for drugs in these systems are found in drug monographs and in the indexes to Analytical Data in Part 3; they are also included in the systems for specific groups of drugs that follow. The search window should be ±50 RIs if hydrocarbons are used to calculate RI, or ±30 RIs if a reference drug mixture is used for the RI calculation.