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#314 Analytical Chemistry Complete

Acetaminophen assay (USP), tablet batch 04-22

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HPLC acetaminophen reverse-phase USP-assay pharma-QC

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This template captures the USP method for acetaminophen (paracetamol) quantification in formulated immediate-release tablets, with caffeine as internal standard.

Goal: determine the acetaminophen content in tablet batch 04-22 (label claim 500 mg/tablet) against a USP Reference Standard, with system-suitability criteria met before and after the sample sequence.

Method overview: Tablets are crushed and a portion equivalent to ~25 mg acetaminophen is dissolved in mobile phase, sonicated, filtered, and diluted to volume. The mobile phase is 80% 50 mM phosphate buffer (pH 4.7) + 20% methanol, run isocratic at 1.5 mL/min through a C18 column (4.6 × 150 mm, 5 µm) at 25°C, with UV detection at 254 nm. A 20 µL injection volume is used. Sample acetaminophen content is calculated by comparison to a USP Reference Standard calibration curve, with caffeine internal standard normalization.

Expected: Acetaminophen retention time 1.9 ± 0.1 min, caffeine retention time 4.4 ± 0.1 min. System suitability: tailing factor ≤ 1.5, theoretical plates ≥ 2000, replicate injection RSD ≤ 2%. Tablet assay result within USP range (90-110% of label claim).

Last saved: 34 seconds ago

12 fields loaded from USP Acetaminophen Assay (Method validated 2024-09)

Method

Column

text

C18, 4.6 × 150 mm, 5 µm

Mobile phase

text

80% 50 mM PO₄ pH 4.7 / 20% MeOH

Flow rate

number

1.5mL/min

Detection

number

254nm

Injection volume

number

20µL

Sample

Analyte

text

Acetaminophen

Sample matrix

select

tablet (IR), tablet (ER), capsule, oral suspension

Reference standard

text

USP Ref Std, lot M0F347

Internal standard

text

Caffeine, 0.5 mg/mL

System Suitability

Tailing factor

number

1.08

Theoretical plates

number

6420

Replicate RSD

number

0.42%

method_v3.pdf

286 KB

chromatogram_sequence.pdf

1.4 MB

integration_results.xlsx

64 KB

01

Prepare mobile phase: 800 mL 50 mM phosphate buffer pH 4.7 + 200 mL MeOH, degas

Sparged 5 min He

0:30

02

Equilibrate column at 1.5 mL/min until stable baseline

0:30

03

Run system suitability: 5 replicate injections of working standard

RSD 0.42% · tailing 1.08

0:40

04

Prepare 5-point standard curve from USP Ref Std (50-150 µg/mL APAP)

0:25

05

Crush and weigh 20 tablets, mill to fine powder, weigh portion → 25 mg APAP

Avg tablet wt 612.4 mg

0:25

06

Dissolve in 50 mL mobile phase, sonicate 10 min, filter 0.45 µm

0:15

07

Inject standard curve in order: 50, 75, 100, 125, 150 µg/mL APAP

0:40

08

Inject samples (triplicate) bracketed by midpoint standard

0:60

09

Inject post-run system suitability replicate set

Confirms no column drift

0:30

10

Integrate peaks, calculate assay result, write summary

502.4 mg/tablet (100.5%)

0:30

	A	B	C	D	E	F	G	H
1	Inj #	Sample	RT APAP (min)	Peak area APAP	RT IS (min)	Peak area IS	APAP (mg/tab)	Notes
2	1	STD-1 (50 µg/mL)	1.91	161,742	4.41	167,408	—	STD curve
3	2	STD-3 (100 µg/mL)	1.92	322,455	4.42	167,221	—	STD curve
4	3	STD-5 (150 µg/mL)	1.91	484,128	4.40	167,512	—	STD curve
5	4	Sample-A rep 1	1.91	485,212	4.41	167,408	502.4	Within USP
6	5	Sample-A rep 2	1.91	483,914	4.41	167,331	501.1	Within USP
7	6	Sample-A rep 3	1.92	486,201	4.40	167,524	503.4	Within USP
8	7	STD-3 mid-bracket	1.92	321,894	4.41	167,247	—	Within ±2%

EXP-2026-0422 Tablet batch 04-22 receipt + sampling F. Mendes Apr 22

EXP-2024-0915 Method validation (precision, accuracy, range) F. Mendes Sep 15 2024

MTH-0091 USP Acetaminophen Tablets Assay Method v3 Methods DB Sep 15 2024

INST-0044 HPLC #2 instrument IQ/OQ qualification record Validation Jan 12

SOP-0218 System Suitability Assessment SOP QA Team Feb 8

USP Acetaminophen Reference Standard

1003009 · Lot M0F347

In stock

Caffeine (internal standard)

C0750 · Lot SLBM7138

In stock

Methanol, HPLC grade

34860 · Lot 19E124501

In stock

Potassium phosphate monobasic (KH₂PO₄)

P5655 · Lot SLCK4926

In stock

C18 column (Symmetry C18 4.6×150 5 µm)

WAT054275 · Col SN 0334121

In stock

0.45 µm PVDF syringe filters

SLHV033RS · Lot R3FA94512

Low

USP Ref Std aliquots (25 mg)

4 °C · QC Reagent Fridge · Shelf 1 · Box RSAM-04

×8

Prepared mobile phase (1 L)

Bench A · MP cabinet · Labelled 04-22

×1

Visibility

QC LabF. Mendes

Can write

F. MendesM. Hartmann

Color

Stroke width

Eraser

Use Ctrl + click to add text

{
    "experiment": {
        "id": 314,
        "custom_id": "HPLC-APAP-2026-04",
        "title": "Acetaminophen assay (USP), tablet batch 04-22",
        "template": "HPLC",
        "template_version": "v3.2",
        "category": "Analytical Chemistry",
        "status": "Complete",
        "started_on": "2026-06-06",
        "tags": [
            "HPLC",
            "acetaminophen",
            "reverse-phase",
            "USP-assay",
            "pharma-QC"
        ]
    },
    "main_text": "This template captures the USP method for acetaminophen (paracetamol) quantification in formulated immediate-release tablets, with caffeine as internal standard.\nGoal: determine the acetaminophen content in tablet batch 04-22 (label claim 500 mg/tablet) against a USP Reference Standard, with system-suitability criteria met before and after the sample sequence.\nMethod overview: Tablets are crushed and a portion equivalent to ~25 mg acetaminophen is dissolved in mobile phase, sonicated, filtered, and diluted to volume. The mobile phase is 80% 50 mM phosphate buffer (pH 4.7) + 20% methanol, run isocratic at 1.5 mL/min through a C18 column (4.6 × 150 mm, 5 µm) at 25°C, with UV detection at 254 nm. A 20 µL injection volume is used. Sample acetaminophen content is calculated by comparison to a USP Reference Standard calibration curve, with caffeine internal standard normalization.\nExpected: Acetaminophen retention time 1.9 ± 0.1 min, caffeine retention time 4.4 ± 0.1 min. System suitability: tailing factor ≤ 1.5, theoretical plates ≥ 2000, replicate injection RSD ≤ 2%. Tablet assay result within USP range (90-110% of label claim).",
    "extra_fields": {
        "Method": {
            "Column": {
                "type": "text",
                "value": "C18, 4.6 × 150 mm, 5 µm"
            },
            "Mobile phase": {
                "type": "text",
                "value": "80% 50 mM PO₄ pH 4.7 / 20% MeOH"
            },
            "Flow rate": {
                "type": "number",
                "value": "1.5 mL/min"
            },
            "Detection": {
                "type": "number",
                "value": "254 nm"
            },
            "Injection volume": {
                "type": "number",
                "value": "20 µL"
            }
        },
        "Sample": {
            "Analyte": {
                "type": "text",
                "value": "Acetaminophen"
            },
            "Sample matrix": {
                "type": "select",
                "value": "tablet (IR), tablet (ER), capsule, oral suspension"
            },
            "Reference standard": {
                "type": "text",
                "value": "USP Ref Std, lot M0F347"
            },
            "Internal standard": {
                "type": "text",
                "value": "Caffeine, 0.5 mg/mL"
            }
        },
        "System Suitability": {
            "Tailing factor": {
                "type": "number",
                "value": "1.08"
            },
            "Theoretical plates": {
                "type": "number",
                "value": "6420"
            },
            "Replicate RSD": {
                "type": "number",
                "value": "0.42 %"
            }
        }
    },
    "attached_files": [
        {
            "filename": "method_v3.pdf",
            "type": "pdf",
            "size": "286 KB"
        },
        {
            "filename": "chromatogram_sequence.pdf",
            "type": "pdf",
            "size": "1.4 MB"
        },
        {
            "filename": "integration_results.xlsx",
            "type": "xlsx",
            "size": "64 KB"
        }
    ],
    "steps": [
        {
            "n": 1,
            "description": "Prepare mobile phase: 800 mL 50 mM phosphate buffer pH 4.7 + 200 mL MeOH, degas",
            "duration": "0:30",
            "complete": true,
            "note": "Sparged 5 min He"
        },
        {
            "n": 2,
            "description": "Equilibrate column at 1.5 mL/min until stable baseline",
            "duration": "0:30",
            "complete": true
        },
        {
            "n": 3,
            "description": "Run system suitability: 5 replicate injections of working standard",
            "duration": "0:40",
            "complete": true,
            "note": "RSD 0.42% · tailing 1.08"
        },
        {
            "n": 4,
            "description": "Prepare 5-point standard curve from USP Ref Std (50-150 µg/mL APAP)",
            "duration": "0:25",
            "complete": true
        },
        {
            "n": 5,
            "description": "Crush and weigh 20 tablets, mill to fine powder, weigh portion → 25 mg APAP",
            "duration": "0:25",
            "complete": true,
            "note": "Avg tablet wt 612.4 mg"
        },
        {
            "n": 6,
            "description": "Dissolve in 50 mL mobile phase, sonicate 10 min, filter 0.45 µm",
            "duration": "0:15",
            "complete": true
        },
        {
            "n": 7,
            "description": "Inject standard curve in order: 50, 75, 100, 125, 150 µg/mL APAP",
            "duration": "0:40",
            "complete": true
        },
        {
            "n": 8,
            "description": "Inject samples (triplicate) bracketed by midpoint standard",
            "duration": "0:60",
            "complete": true
        },
        {
            "n": 9,
            "description": "Inject post-run system suitability replicate set",
            "duration": "0:30",
            "complete": true,
            "note": "Confirms no column drift"
        },
        {
            "n": 10,
            "description": "Integrate peaks, calculate assay result, write summary",
            "duration": "0:30",
            "complete": true,
            "note": "502.4 mg/tablet (100.5%)"
        }
    ],
    "spreadsheet": [
        [
            "Inj #",
            "Sample",
            "RT APAP (min)",
            "Peak area APAP",
            "RT IS (min)",
            "Peak area IS",
            "APAP (mg/tab)",
            "Notes"
        ],
        [
            "1",
            "STD-1 (50 µg/mL)",
            "1.91",
            "161,742",
            "4.41",
            "167,408",
            "—",
            "STD curve"
        ],
        [
            "2",
            "STD-3 (100 µg/mL)",
            "1.92",
            "322,455",
            "4.42",
            "167,221",
            "—",
            "STD curve"
        ],
        [
            "3",
            "STD-5 (150 µg/mL)",
            "1.91",
            "484,128",
            "4.40",
            "167,512",
            "—",
            "STD curve"
        ],
        [
            "4",
            "Sample-A rep 1",
            "1.91",
            "485,212",
            "4.41",
            "167,408",
            "502.4",
            "Within USP"
        ],
        [
            "5",
            "Sample-A rep 2",
            "1.91",
            "483,914",
            "4.41",
            "167,331",
            "501.1",
            "Within USP"
        ],
        [
            "6",
            "Sample-A rep 3",
            "1.92",
            "486,201",
            "4.40",
            "167,524",
            "503.4",
            "Within USP"
        ],
        [
            "7",
            "STD-3 mid-bracket",
            "1.92",
            "321,894",
            "4.41",
            "167,247",
            "—",
            "Within ±2%"
        ]
    ],
    "links": {
        "experiments": [
            {
                "ref_id": "EXP-2026-0422",
                "title": "Tablet batch 04-22 receipt + sampling",
                "owner": "F. Mendes",
                "date": "Apr 22"
            },
            {
                "ref_id": "EXP-2024-0915",
                "title": "Method validation (precision, accuracy, range)",
                "owner": "F. Mendes",
                "date": "Sep 15 2024"
            }
        ],
        "resources": [
            {
                "ref_id": "MTH-0091",
                "title": "USP Acetaminophen Tablets Assay Method v3",
                "owner": "Methods DB",
                "date": "Sep 15 2024"
            },
            {
                "ref_id": "INST-0044",
                "title": "HPLC #2 instrument IQ/OQ qualification record",
                "owner": "Validation",
                "date": "Jan 12"
            },
            {
                "ref_id": "SOP-0218",
                "title": "System Suitability Assessment SOP",
                "owner": "QA Team",
                "date": "Feb 8"
            }
        ]
    },
    "compounds": [
        {
            "name": "USP Acetaminophen Reference Standard",
            "catalog_lot": "1003009 · Lot M0F347",
            "stock_status": "In stock"
        },
        {
            "name": "Caffeine (internal standard)",
            "catalog_lot": "C0750 · Lot SLBM7138",
            "stock_status": "In stock"
        },
        {
            "name": "Methanol, HPLC grade",
            "catalog_lot": "34860 · Lot 19E124501",
            "stock_status": "In stock"
        },
        {
            "name": "Potassium phosphate monobasic (KH₂PO₄)",
            "catalog_lot": "P5655 · Lot SLCK4926",
            "stock_status": "In stock"
        },
        {
            "name": "C18 column (Symmetry C18 4.6×150 5 µm)",
            "catalog_lot": "WAT054275 · Col SN 0334121",
            "stock_status": "In stock"
        },
        {
            "name": "0.45 µm PVDF syringe filters",
            "catalog_lot": "SLHV033RS · Lot R3FA94512",
            "stock_status": "Low"
        }
    ],
    "storage": [
        {
            "name": "USP Ref Std aliquots (25 mg)",
            "location": "4 °C · QC Reagent Fridge · Shelf 1 · Box RSAM-04",
            "count": "×8"
        },
        {
            "name": "Prepared mobile phase (1 L)",
            "location": "Bench A · MP cabinet · Labelled 04-22",
            "count": "×1"
        }
    ],
    "permissions": {
        "visibility": [
            {
                "type": "Team",
                "name": "QC Lab"
            },
            {
                "type": "Person",
                "name": "F. Mendes"
            }
        ],
        "can_write": [
            {
                "type": "Person",
                "name": "F. Mendes"
            },
            {
                "type": "Person",
                "name": "M. Hartmann"
            }
        ]
    }
}