# SOP — Estradiol Valerate Assay by RP-HPLC-UV on Agilent 1100

**Scope:** a replicable, audit-survivable method for **identity + potency** of estradiol valerate (EV) in oil-based injectables on a refurbished Agilent 1100. Target readership: a DIY/community lab operator with the instrument in hand, a USP EV Reference Standard, a C18 column, and the intent to run method validation before reporting numbers.

**Last updated:** 2026-04-23
**Status:** deep-dive SOP. Not a substitute for a USP monograph. Community practice is flagged throughout.
**Related Round-1 docs:** [`../02-analytical-chemistry/hplc.md`](../02-analytical-chemistry/hplc.md) (general theory), [`../04-equipment-market/hplc-used-prices.md`](../04-equipment-market/hplc-used-prices.md) (instrument market), [`../06-reference-standards/overview.md`](../06-reference-standards/overview.md) (RS chain of trust), [`../08-interpretation-qc/oil-formulation-sample-prep.md`](../08-interpretation-qc/oil-formulation-sample-prep.md) (oil prep), [`../05-home-lab-setup/failure-modes.md`](../05-home-lab-setup/failure-modes.md) (general failure catalog).

---

## Key takeaways

1. **A refurbished Agilent 1100 with G1311A quaternary pump + G1313A ALS + G1316A column compartment + G1315B DAD is the minimum instrument that produces audit-survivable EV potency + identity data.** A VWD (G1314A/B) is cheaper but gives you retention-time matching only — DAD spectral match is what distinguishes EV from other estradiol esters at the same retention time.
2. **Isocratic methanol/water 80:20 on a 4.6×150 mm, 5 µm L1 C18 column at 1.0 mL/min, UV 280 nm, 15-min run** is the canonical estradiol-ester method. It resolves EV from free estradiol and estrone cleanly and is USP <621>-adjustable without revalidation within defined limits.
3. **Oil-based HRT must be diluted into a mobile-phase-compatible solvent before injection** — never inject neat oil. Dilute-and-shoot in methanol is fine for MCT/grapeseed; ACN freeze-precipitation is required for castor oil and for any LC-MS follow-up.
4. **System suitability — 5 replicate injections of a mid-curve standard with RSD ≤ 2.0%, N ≥ 2000, T ≤ 2.0, Rs ≥ 1.5 — must pass before any sample is reported.** This is USP <621> non-negotiable. Skipping it means your numbers cannot be defended.
5. **Identity acceptance = retention time within ±2% of standard AND DAD spectral match factor ≥ 0.998. Potency acceptance = 90.0–110.0% of label claim** (USP general monograph limit for injection preparations). Bracket every 10–20 samples with a standard to monitor drift.

---

## 1. Instrument setup — Agilent 1100

### 1.1 Required modules

The 1100 is modular; a refurb auction lot may be missing pieces. The minimum viable EV-assay stack:

| Module | Part | Function | Notes |
|---|---|---|---|
| Solvent degasser | **G1322A** | In-line vacuum degasser, 4-channel | Not strictly required if you off-line helium-sparge or sonicate, but sparing it saves a labor step per run. |
| Pump | **G1311A** quaternary *or* **G1312A** binary | Delivers mobile phase | See §1.2 below — choose carefully. |
| Autosampler (ALS) | **G1313A** | 100-vial standard ALS | **G1329A** is the thermostatted variant — useful if your standards degrade at room temp. |
| Column compartment | **G1316A** thermostatted column compartment (TCC) | Holds column at 25–35 °C | Non-thermostatted compartments exist and are cheaper but make retention time control worse. Insist on the G1316A. |
| Detector | **G1315B DAD** *or* **G1314A/B VWD** | UV/DAD 190–950 nm / single-wavelength VWD | See §1.3. |

**Claim:** Agilent 1100 module codes G1311A (quaternary pump), G1312A (binary pump), G1313A (standard ALS), G1314A/B (VWD), G1315B (DAD), G1316A (thermostatted column compartment), G1322A (four-channel in-line degasser) are the canonical designations used in refurb listings.
**Confidence:** C2
**Source:** LabX listings cross-referencing modules (https://www.labx.com/item/agilent-hp-1100-hplc-chemstation-g1322a-g1311a-g1315b/14519844), Agilent 1100 Quaternary Pump Reference Manual (https://conquerscientific.com/wp-content/uploads/2022/10/agilent-1100-series-quaternary-pump-g1311a_manual.pdf)
**Date checked:** 2026-04-23

### 1.2 Quaternary (G1311A) vs binary (G1312A) pump — which to buy

**For HRT isocratic work, the G1311A quaternary is the correct choice.** Reasoning:

- Isocratic EV assay uses one mobile phase. Either pump handles that.
- The quaternary mixes in the low-pressure side (before the pump), so on-the-fly ratio changes (e.g. 80:20 vs 75:25 for method development) are click-and-run. The binary mixes high-pressure, which gives better reproducibility for fast gradients but is more hardware to fail.
- The G1312A binary is *better* for gradient work (related-substances impurity profiling, USP-style system suitability of mixed estrogens). If you intend to do related-substances impurity work later, buy binary. If you are strictly running identity + potency, quaternary is the pragmatic buy.
- Used prices cluster similarly; the G1312A tends to run slightly higher at auction.

**Community practice:** most refurb lots ship as G1311A quaternary because that was Agilent's default config for the 1100 era. If you see a G1312A-only listing with no matching ChemStation config, verify the pump was actually paired with DAD+ALS before buying.

### 1.3 VWD vs DAD — buy the DAD

- **VWD (G1314A/B)** = single-wavelength UV. Cheaper. Gives you a chromatogram at 280 nm and nothing else. Identity = retention time only, which cannot distinguish EV from estradiol enanthate (EEn) or other C17-esters that coelute on short columns.
- **DAD (G1315B)** = diode array, 190–950 nm, full spectrum per data point. Identity = retention time + UV spectral match. ChemStation's peak-purity and spectral-match tools (Library-Match, Peak Purity Index) depend on DAD. **For EV in particular — where the wrong-ester failure mode is common in gray-market product — DAD is the difference between a method that catches a mis-ester and one that silently passes it.**

Used-market bands from [`../04-equipment-market/hplc-used-prices.md`](../04-equipment-market/hplc-used-prices.md): Agilent 1100 + VWD refurb ~$12,390; Agilent 1100 + DAD refurb ~$15,530. The DAD premium is ~$3k on the refurb tier and closer to $4–6k at the eBay/as-is tier. **Pay the DAD premium.** A mis-identified EV that was actually EEn at the same label dose is a credible gray-market failure and DAD is what catches it.

### 1.4 ChemStation revision and PC

**ChemStation Rev. B.04.03 (SP1 or SP2) is the last 1100-compatible revision Agilent released.** Per Agilent's lifecycle notice, the Agilent 1100 series reaches end-of-guaranteed-support 2026-01-31 — so as of this SOP (2026-04-23) the 1100 is officially unsupported. Driver downloads and the B.04.03 installer remain obtainable through Agilent community forums and refurb vendors; Agilent support engineers will no longer answer 1100-specific tickets.

- **Operating system:** B.04.03 is certified on Windows 7 (32-bit), Windows Vista SP2, Windows XP SP3. Community reports that SP2 installs work on Windows 10 32-bit with compatibility-mode tweaks (unsupported by Agilent).
- **PC interface — the critical check.** Early 1100 modules communicated over **GPIB/HPIB** (requires an 82350B GPIB card in the PC — still available on eBay ~$150). Later 1100 modules shipped with built-in **LAN** interfaces; LAN came in mid-lifecycle (circa 2004 for the detectors, later for pumps). Parallel/IEEE-1284 connections existed on the very earliest 1100 modules. **Check the serial number + back-panel connector of every module in the lot before buying.** A LAN-only refurb can go straight onto a hub-and-switch; a GPIB refurb needs the 82350B card plus a PC with a PCI/PCIe slot that will accept it.

**Claim:** ChemStation B.04.03 is the last revision supporting Agilent 1100 modules; Agilent 1100 end-of-guaranteed-support date is 2026-01-31.
**Confidence:** C2
**Source:** Agilent community forum (https://community.agilent.com/technical/software/f/forum/3667/chemstation-b-04-03), Chromatography Forum discussion (https://www.chromforum.org/viewtopic.php?t=26656)
**Date checked:** 2026-04-23
**Notes:** EOL date has been stated in several community forum threads; the authoritative Agilent product-discontinuation announcement is not on a public URL that I could reach today. Verify against your refurb vendor's documentation before committing.

### 1.5 Install checklist

1. Lab bench: stable, vibration-damped, ≥120 cm wide for the stack + PC + printer.
2. Mains: 115 V or 230 V (check module labels — region-specific PSUs), dedicated 15 A circuit preferred (the pump + DAD lamp draws ~5 A on startup).
3. Solvent reservoirs: 1 L amber bottles with GL45 caps, PTFE inlet filter, on a rack above the instrument.
4. Waste: 4 L amber jerrycan with labeled "HPLC waste — methanol/water" sticker. Acetonitrile waste segregated from chlorinated solvents.
5. PC: ChemStation-installed Windows 7 (32-bit) machine, or a validated refurb. GPIB or LAN cable per your module interface.
6. First power-up: allow 30 min for D2 lamp warm-up before any quantitative run (lamp drift is the dominant source of absolute-response drift in hour 1).

---

## 2. Column selection

The USP designation to look for on the CoA is **L1** (octadecylsilane chemically bonded to porous silica particles). Any L1 column that meets the dimensional tolerances is nominally USP-interchangeable without revalidation per USP <621> (see §7 below).

**Two recommended columns** (either is fine; choice is availability + budget):

### 2.1 Waters Symmetry C18, 100 Å, 5 µm, 4.6 × 150 mm

- **Part number: WAT045905** (Waters catalog designation for the 4.6 × 150 mm configuration)
- Pore size 100 Å, particle 5 µm, fully porous, endcapped, low-silanol
- pH range 2–8, max pressure ~6000 psi
- USP L1

**Claim:** Waters Symmetry C18 100 Å, 5 µm, 4.6 × 150 mm is Waters catalog WAT045905; the 250 mm length equivalent is WAT054275.
**Confidence:** C2
**Source:** Waters catalog pages https://www.waters.com/nextgen/us/en/shop/columns/wat045905-symmetry-c18-column-100a-5--m-46-mm-x-150-mm-1-pk.html ; https://www.waters.com/nextgen/us/en/shop/columns/wat054275-symmetry-c18-column-100a-5--m-46-mm-x-250-mm-1-pk.html
**Date checked:** 2026-04-23
**Notes:** Direct fetch timed out on the 150 mm SKU; SKU confirmed via Waters search result URL and cross-reference to the adjacent 250 mm SKU. Verify on waters.com before ordering.

### 2.2 Agilent ZORBAX Eclipse Plus C18, 95 Å, 5 µm, 4.6 × 150 mm

- **Part number: 959993-902** (Agilent catalog designation for 4.6 × 150 mm, 5 µm, 95 Å)
- Density-bonded, double-endcapped silica — low silanol activity
- pH range 2–9, max pressure ~600 bar
- USP L1

**Claim:** Agilent ZORBAX Eclipse Plus C18 4.6 × 150 mm, 5 µm is Agilent part 959993-902 (the 4.6 × 100 mm equivalent in 3.5 µm is 959961-902 — not the same part).
**Confidence:** C2
**Source:** https://www.netascientific.com/lc-c1-columns/959993-902 ; https://chromtech.com/zorbax-eclipse-plus/
**Date checked:** 2026-04-23
**Notes:** Eclipse Plus 4.6 × 150 mm 5 µm is widely stocked. Similar generic L1 columns (Phenomenex Luna C18(2), Restek Raptor C18, Thermo Hypersil Gold C18) will give equivalent selectivity; the published validation data for EV is mostly on Zorbax and XBridge.

### 2.3 Guard column

- **Phenomenex SecurityGuard C18, 4 × 3.0 mm ID, part AJ0-4287** (10-pack cartridge) plus **KJ0-4282 holder**. Compatible with all 3.2–8.0 mm ID analytical columns (not core-shell/UHPLC).
- Pressure-rated 3500 psi (241 bar) — below analytical-column rating; acceptable because your method is ≤150 bar.

**Claim:** Phenomenex SecurityGuard AJ0-4287 is the standard C18 guard-cartridge pack (10/pk), 4 × 3.0 mm ID, for analytical columns 3.2–8.0 mm ID; requires holder KJ0-4282; pressure rating 3500 psi.
**Confidence:** C1
**Source:** https://www.phenomenex.com/part?partNo=AJ0-4287
**Date checked:** 2026-04-23

**Replacement schedule:** new guard cartridge every 50–100 oil-matrix injections, or when back-pressure creeps more than 20% above clean baseline, whichever is first. For clean aqueous standards, guards last 200+ injections.

### 2.4 USP estradiol valerate monograph — what the published literature says the monograph requires

The current USP-NF Estradiol Valerate monograph is paywalled at https://online.uspnf.com. The following parameters are **reconstructed from secondary sources** — do not quote as if you read the live monograph:

- Column: L1 (C18) or L7 (C8), 4.6 × 150 mm, 5 µm typical
- Mobile phase: methanol/water (approximately 80:20, adjustable under USP <621>)
- Detection: UV, 280 nm (or 220 nm for higher sensitivity)
- Flow rate: ~1.0 mL/min
- System suitability: tailing ≤ 2.0, plates ≥ 2000, replicate RSD ≤ 2.0%

**Claim:** The USP Estradiol Valerate monograph parameters above are reconstructed from peer-reviewed HPLC method papers (Bentham Science, Scholars Research Library) citing USP; from the Waters application note 720001985EN on XBridge C18 USP Estradiol analysis; and from USP general chapter <621> harmonized-text PDF. Primary USP-NF text not verified in this pass.
**Confidence:** C3 (method parameters are well-supported by independent literature; specific monograph text is not)
**Source:** https://www.scholarsresearchlibrary.com/articles/validated-rphplc-method-for-the-determination-of-estradiol-valerate-inbulk-and-pharmaceutical-formulations.pdf ; https://www.waters.com/nextgen/us/en/library/application-notes/2007/analysis-of-estradiol-usp-method-using-xbridge-c18.html ; https://www.usp.org/sites/default/files/usp/document/harmonization/gen-chapter/harmonization-november-2021-m99380.pdf
**Date checked:** 2026-04-23
**Notes:** If you need the literal monograph text for audit, buy a USP-NF subscription ($1,685/year individual tier as of 2026) or pull the monograph from a university library.

---

## 3. Mobile phase

### 3.1 Option A: Methanol/water 80:20 (v/v) — USP-style, the default

Simplest. Meets the reconstructed monograph. Works across all L1 columns without modification.

**Prep (1 L):**
1. Measure 800 mL HPLC-grade methanol (Fisher A4524 $183/4 L, Lab Alley equivalent — see [`../04-equipment-market/consumables-costs.md`](../04-equipment-market/consumables-costs.md)) into a 1 L graduated cylinder.
2. Add 200 mL HPLC-grade water (18.2 MΩ Milli-Q fresh, *not* RO, *not* WFI unless specified LC-grade).
3. Transfer to a 1 L amber solvent bottle. Cap with the PTFE-lined cap.
4. Degas: sonicate 10 min in a benchtop sonicator, *or* rely on the in-line G1322A degasser (use both if the bottle is fresh — G1322A alone is not enough for a freshly-mixed bottle).

**Filtration** — optional for premixed methanol/water because both solvents are HPLC-grade by CoA. If you prepare from non-HPLC water (e.g., house-distilled), filter the aqueous portion through 0.45 µm nylon before mixing; nylon is correct for aqueous. Do not filter pure methanol through nylon — use PTFE if you must filter the organic.

**Shelf life:** 2 weeks at room temperature in amber bottle. Discard after 2 weeks or at the first sign of microbial growth (cloudiness, floaters) — methanol/water 80:20 is microbial-hostile but not sterile.

### 3.2 Option B: Acetonitrile/phosphate buffer pH 3 — community alternative

Sharper peaks, less silanol-related tailing on older columns, lower back-pressure than MeOH/water (ACN/water is ~40% less viscous at 80:20 than MeOH/water at 80:20). Trade-off: ACN shortage + price volatility post-2025 supply disruption (Lab Alley $188–$225/4 L as of 2026-04-22), plus the buffer adds prep complexity.

**Recipe (1 L):**
1. Weigh 1.36 g KH₂PO₄ (monobasic potassium phosphate, ACS reagent grade) into a 200 mL beaker.
2. Dissolve in ~150 mL Milli-Q water, adjust to pH 3.0 with dilute orthophosphoric acid (drop-wise, pH meter calibrated that day).
3. Transfer to 1 L volumetric; add 150 mL more Milli-Q to bring aqueous to 200 mL total.
4. Add 800 mL HPLC-grade acetonitrile.
5. Filter through 0.45 µm nylon (aqueous-compatible) into amber solvent bottle.
6. Degas: sonicate 10 min.

**Community practice:** 25 mM phosphate at pH 3 is common but not in the reconstructed USP monograph for EV — flag as "community practice, validate on your system." Documented in peer-reviewed method papers for related-substances work but not for the USP assay.

### 3.3 Which to choose for a home lab

- If you are running a single-analyte EV potency method and nothing else: **Option A (MeOH/water 80:20).** Fewer failure modes.
- If you are also running testosterone esters, progesterone, spironolactone, or doing related-substances work: **Option B (ACN + phosphate).** Better peak shape across a wider analyte range. Pay for it.

---

## 4. Chromatographic conditions (isocratic)

| Parameter | Value | Notes |
|---|---|---|
| Column | L1 C18, 4.6 × 150 mm, 5 µm (see §2) | Guard cartridge in-line |
| Mobile phase | MeOH/H₂O 80:20 (v/v), isocratic | Or Option B, §3.2 |
| Flow rate | **1.0 mL/min** | USP <621>-allowable adjustment: ±50% (0.5–1.5 mL/min) |
| Column temperature | **30 °C** (± 2 °C) | Via G1316A TCC |
| Injection volume | **10 µL** | For 40 mg/mL vial diluted 1:1000 — middle of linear range |
| Detection wavelength | **280 nm** (primary) | DAD secondary: record 220 nm + full 200–400 nm spectrum |
| Bandwidth | 4 nm | ChemStation DAD default |
| Reference wavelength | 360 nm, bandwidth 100 nm | Baseline subtraction |
| Total run time | **15 min** | EV elutes ~8.2 min; allow 1.8× buffer for matrix components |
| Re-equilibration | None (isocratic) | 10 column volumes between mobile-phase changes |

**Expected retention times** (mobile phase A, 30 °C, 1.0 mL/min, 4.6 × 150 mm L1):

| Analyte | tR (min) | Notes |
|---|---|---|
| Dead time (t₀) | ~1.8 | Acetone or uracil marker |
| Free estradiol (E2) | ~3.5 | Hydrolysis impurity |
| Estrone (E1) | ~4.8 | Oxidation impurity |
| Ethinyl estradiol (EE — optional IS) | ~4.2 | Alternative IS |
| **Estradiol valerate (EV)** | **~8.2** | Target |
| Estradiol enanthate (EEn) | ~9.6 | Wrong-ester check |
| Estradiol cypionate (EC) | ~11.8 | Can be used as IS (see §6.3) |
| Testosterone enanthate (TE — optional IS) | ~13.5 | Alternative IS |

Retention times are **approximate, column-and-column-age-dependent**. They will drift with aging. System suitability at the start of each run establishes the accepted RT window for that day.

---

## 5. Sample preparation (oil injectables)

Detailed theory is in [`../08-interpretation-qc/oil-formulation-sample-prep.md`](../08-interpretation-qc/oil-formulation-sample-prep.md). This section specifies the *exact* operations for EV.

### 5.1 Decision tree

| Vial carrier | Prep method | Rationale |
|---|---|---|
| MCT (medium-chain triglycerides) | **Dilute-and-shoot in methanol** (§5.2) | Carrier dissolves in MeOH, minimal column fouling |
| Grapeseed | Dilute-and-shoot in methanol | Similar to MCT |
| Cottonseed, sesame | Dilute-and-shoot in methanol; optional LLE | Workable either way |
| Castor | **LLE with ACN freeze-precipitation** (§5.3) | Highly viscous, non-polar; direct dilution fouls |
| Unknown carrier | LLE to be safe | |
| Any carrier going to LC-MS confirmation | **LLE mandatory** | Oil in ion source = catastrophic |

### 5.2 Dilute-and-shoot (MCT, grapeseed, most HRT)

For a 40 mg/mL EV vial, target final 40 µg/mL in injection vial (inject 10 µL = 400 ng on column — middle of calibration curve).

1. Equilibrate a new USP-certified 10 mL volumetric flask to room temperature.
2. Using a calibrated positive-displacement pipette (viscous-liquid compatible — Gilson MICROMAN E or equivalent), transfer **100 µL oil injectable** into the flask.
3. Add ~5 mL HPLC-grade methanol; cap, shake, sonicate 5 min.
4. Dilute to 10.0 mL with methanol; mix by inversion 10×.
5. This gives 400 µg/mL — still 10× too concentrated. Transfer 1.00 mL to a second 10 mL volumetric, dilute to 10.0 mL with methanol → 40 µg/mL final.
6. Filter ~1 mL through 0.22 µm PTFE syringe filter (13 mm) into a 2 mL amber HPLC autosampler vial. Cap with pre-slit septum.
7. Label: lot, dilution factor (1:1000), date, operator initials.

**Total dilution factor: 1:1000.** Keep this constant across samples and standards for direct comparability.

### 5.3 LLE with ACN freeze-precipitation (castor, LC-MS follow-up)

1. Transfer **100 µL oil injectable** into a 2 mL microcentrifuge tube (polypropylene, solvent-resistant).
2. Add **1.0 mL HPLC-grade acetonitrile**. Cap. Vortex 30 s.
3. Freeze at **−20 °C for 60 min**. Oil phase congeals at the bottom; ACN supernatant clarifies.
4. Centrifuge 14,000 rpm (21,000 × g) for 5 min at 4 °C.
5. Transfer **900 µL of clear supernatant** to a clean 2 mL tube.
6. Further dilute 1:10 in mobile phase (100 µL supernatant + 900 µL mobile phase) to hit ~40 µg/mL.
7. Filter 0.22 µm PTFE into HPLC vial.
8. Label with prep method, dilution factor (1:111, since 100 µL in 1.1 mL ACN + water workup = ~10-fold in ACN, then 10× in diluent).

**Recovery validation required** per [`../08-interpretation-qc/oil-formulation-sample-prep.md`](../08-interpretation-qc/oil-formulation-sample-prep.md): spike a blank carrier oil with USP EV RS at 40 mg/mL, run the prep, quantify against a standard curve in pure ACN. Target recovery 95–105%.

### 5.4 Diluent choice and solvent-mismatch

The injection diluent must be **weaker than** or **equal to** the mobile phase organic content. Mobile phase is 80% methanol; injecting in 100% methanol gives solvent mismatch (sample plug elutes ahead of mobile-phase front → peak fronting/splitting). Two fixes:

- **Dilute in mobile phase itself** (80:20 MeOH/H₂O) for the final dilution step. Oil solubility drops at 80:20 vs 100% MeOH, so the first dilution (step 3 in §5.2) should still use 100% MeOH for oil dispersion; the final 10× dilution is in 80:20.
- **Inject 5 µL instead of 10 µL** — cuts the plug width by half and usually eliminates visible fronting.

**IPA dissolves oil better than methanol** but IPA is much stronger than mobile phase (≈95–100% equivalent), so IPA dilution causes severe solvent mismatch. Community practice: methanol with extended sonication.

---

## 6. Calibration curve

### 6.1 Reference standard

- **USP Estradiol Valerate Reference Standard, catalog # 1254009, 100 mg vial, $297** (USP catalog price 2026-04-22, see [`../_sources/usp-reference-standards-pricing-2026-04-22.md`](../_sources/usp-reference-standards-pricing-2026-04-22.md))
- CAS 979-32-8, non-scheduled (no DEA registration needed)
- Store at 2–8 °C in desiccator. Hygroscopic — open, weigh, reseal within 60 s. Water gain of 0.5% is typical for a slow weigh.
- **Internal standard (IS):** one of
  - Estradiol cypionate (EC, USP #1252003, $277/200 mg) — resolves from EV by ~3.6 min at the conditions in §4; excellent option if your samples never contain EC.
  - Testosterone enanthate (TE) — cheap non-USP RS (~$50 from Sigma PHR-grade), 240 nm co-detection, elutes at ~13.5 min, well-resolved from EV.
  - **Do NOT use deuterated IS** (e.g. estradiol-d4) for UV work — isotopologues don't separate on RP, so the UV detector sees a single combined peak. Deuterated IS is only useful for MS.

### 6.2 Primary stock preparation

1. On a **0.01 mg analytical balance** (e.g., Mettler XPE105 or refurb equivalent), weigh **≥10 mg** USP EV RS into a 10 mL volumetric flask. Record to 4 decimal places (e.g., 10.23 mg).
2. Dissolve + dilute to 10.0 mL with methanol. Stock concentration = 1.023 mg/mL = 1023 µg/mL.
3. Record mass, flask volume, date, lot number of RS. This is your primary stock.
4. Store at −20 °C in 2 mL aliquots. Shelf life 3 months from prep (community-practice assumption; confirm via monthly re-verification against a fresh prep).

### 6.3 Working calibration standards

Target the sample dilution (1:1000 → 40 µg/mL) in the middle of the curve. Curve at 50%, 80%, 100%, 120%, 150% of nominal:

| Level | Target (µg/mL) | Prep |
|---|---|---|
| 50% | 20 µg/mL | 196 µL stock + 9.8 mL methanol → 10.0 mL |
| 80% | 32 µg/mL | 313 µL stock + 9.687 mL methanol → 10.0 mL |
| 100% | 40 µg/mL | 391 µL stock + 9.609 mL methanol → 10.0 mL |
| 120% | 48 µg/mL | 469 µL stock + 9.531 mL methanol → 10.0 mL |
| 150% | 60 µg/mL | 587 µL stock + 9.413 mL methanol → 10.0 mL |

(Volumes calculated assuming stock = 1023 µg/mL. Recalculate for your actual weighed mass.)

If using IS: add the IS to *all* calibration standards and all samples at a constant concentration (e.g., 20 µg/mL EC or 40 µg/mL TE).

### 6.4 Acceptance of calibration curve

- **Linearity R² ≥ 0.999** across all 5 levels (ICH Q2(R1), USP <1225>)
- **Residuals ≤ ±2.0%** at each level when back-calculated
- **Back-calculate each standard's concentration from the regression**; any level >±2% off target is rejected and re-prepped
- Use **response = area** for external standard; **response = area(EV)/area(IS)** for internal standard

---

## 7. System suitability (USP <621>)

**This must pass before ANY sample result is reportable.** No exceptions.

### 7.1 Criteria

Run **5 replicate injections** of the 100% calibration standard (40 µg/mL EV) at the start of each analytical sequence. Compute:

| Parameter | Criterion | Source |
|---|---|---|
| Replicate RSD of peak area (n=5) | **≤ 2.0%** | USP <621> (general rule) |
| Theoretical plates (N) | **≥ 2000** | Monograph-specific (<621> sets no absolute floor; 2000 is the typical estradiol-ester monograph value) |
| Tailing factor (T, USP method) | **≤ 2.0** | Monograph-specific (<621> default peak-symmetry range is 0.8–1.8) |
| Resolution Rs (EV vs IS, or EV vs nearest related peak) | **≥ 1.5** | USP <621> |
| Retention time reproducibility | RSD ≤ 1.0% | Community practice |
| Baseline drift over 5 injections | ≤ 5% full-scale | Community practice |

**Claim:** USP <621> (harmonized 2022 revision) specifies replicate-injection repeatability RSD ≤ 2.0% (from 5 replicates) as a system-suitability criterion; the chapter itself sets no absolute theoretical-plate (N), tailing (T), or Rs floors — those come from the individual monograph, typically T ≤ 2.0 (general default 0.8–1.8), N ≥ 2000 for isocratic C18 methods, Rs ≥ 1.5 between adjacent peaks. The chapter permits **LC** method adjustments without revalidation within these explicit bounds: **column dimensions: L and ID may be adjusted provided the L/dp ratio remains constant or moves within −25% to +50% of the reference (equivalently, N within −25% to +50%)**; **particle size is NOT freely interchangeable within an L-category — it must satisfy the L/dp rule**; **flow rate ± 50%**; **mobile-phase minor component ± 30% relative, not exceeding ± 10% absolute** (for 80:20 MeOH/water, MeOH may range 74–86%); **pH ± 0.2 of buffered mobile phase**; **column temperature ± 10 °C**; **injection volume adjusted to maintain detector response within linearity**.
**Confidence:** C2
**Source:** https://www.usp.org/sites/default/files/usp/document/harmonization/gen-chapter/harmonization-november-2021-m99380.pdf ; https://www.chromatographyonline.com/view/what-the-new-usp-chapter-621-guidelines-mean-for-your-hplc-analysis
**Date checked:** 2026-04-23
**Round-2 correction (R2-M1):** earlier draft stated "column ± 70%/± 50%" (that is the **GC** rule), "particle size interchangeable within L-category" (not true), and "organic phase ± 2% absolute" (the real rule is ± 30% relative capped at ± 10% absolute). These have been corrected above.

### 7.2 Failure action

- **RSD > 2%:** injection reproducibility problem. Check: ALS needle seat wear, autosampler syringe seal, bubble in pump. Re-inject after degassing.
- **N < 2000:** column efficiency loss. Check: column age (replace if >500 injections), guard cartridge (replace), mobile phase freshness.
- **T > 2.0:** peak tailing. Check: mobile phase pH drift (repeat pH adjustment), silanol activity (replace column), detector bandwidth too wide.
- **Rs < 1.5:** analyte and IS insufficiently resolved. Check: column aged; adjust mobile phase composition within USP <621> limits (e.g., 78:22 MeOH:H₂O for slightly more retention).

### 7.3 Bracketing standards

After passing suitability, bracket samples with standards:
- Sequence: [5× SST] → [100% std] → [10 samples] → [100% std] → [10 samples] → [100% std] → ...
- Allowed drift between bracket standards: ≤ 2.0%
- If drift > 2%, reject all samples between last in-spec bracket and the out-of-spec one; re-run.

---

## 8. Acceptance criteria — identity + potency

### 8.1 Identity

A sample peak is **identified as estradiol valerate** only if ALL of:

1. **Retention time match:** sample peak tR within **±2%** of mean standard tR from the bracketing standards. At tR ≈ 8.2 min, the window is ~8.03–8.36 min.
2. **DAD UV spectral match:** ChemStation spectral comparison against the standard library, **match factor ≥ 0.998** (peak purity reported by ChemStation on a 0–1 scale; threshold corresponds to visually identical spectra). If you only have a VWD, this step cannot be performed — identity is *retention time only*, which is insufficient to distinguish EV from other C17-esters.
3. **No coelution:** ChemStation peak purity index for the analyte peak ≥ 990 (integer 0–1000 scale).

**Optional orthogonal confirmation (L4-level):** LC-MS [M+H]⁺ = 357.2 for EV (molecular formula C₂₃H₃₂O₃, exact mass 356.235). Only relevant if you have LC-MS access.

### 8.2 Potency

- **Acceptance:** 90.0–110.0% of label claim. This is the USP general monograph limit for injection preparations.
- **Report:** "Potency = X.X% of label claim" with 95% confidence interval from the replicate n of the sample (n ≥ 3 injections).
- **Reject:** any sample with potency < 90% or > 110% → flagged FAIL. Re-prep and re-run the sample once; if still out, confirm with a second analyst / second day.

### 8.3 Reject states requiring re-run

- System suitability fails → fix instrument, re-run full sequence
- Bracket standard drift > 2% → reject samples between brackets
- Sample peak tR > ±2% of standard → identity fail; investigate (wrong ester?)
- DAD match < 0.998 → spectral mismatch; investigate (impurity? wrong compound?)
- Peak purity index < 990 → coelution suspected; re-prep, or switch method to gradient to resolve

---

## 9. Troubleshooting

| Symptom | Likely cause | Corrective action |
|---|---|---|
| **Ghost peaks in blank injections** | Mobile phase contamination (bottle, solvent grade), filter contamination, septum contamination | Run 100% methanol flush 20 min at 1.0 mL/min; replace solvent reservoir and filter; switch to LC-MS-grade MeOH if persistent |
| **Baseline drift (monotonic, first 30 min)** | D2 lamp warming up | Allow 30 min lamp-on before quantitative runs; routine |
| **Baseline drift (monotonic, >30 min)** | Column not equilibrated after mobile-phase change | Pump 10+ column volumes (≥25 mL for 4.6 × 150 mm) before next sample |
| **Double peak / shoulder on analyte** | Solvent mismatch (sample diluent stronger than mobile phase) | Dilute final sample in mobile phase not pure methanol; reduce injection volume to 5 µL |
| **Peak tailing (T > 2.0)** | Silanol activity (column aging); pH out of range; dirty column | Replace column; recheck buffer pH; run MeOH/water/IPA cleaning sequence per Agilent SOP |
| **Drifting retention time, day over day** | Column oven not controlling; column aging; mobile phase evaporation from reservoir | Confirm TCC setpoint; cap reservoir with PTFE-lined cap; replace column after 500 injections |
| **Loss of sensitivity (response dropping at fixed concentration)** | D2 lamp decay (1000–2000 h life); dirty flow cell | Replace D2 lamp ($600–800); flush flow cell with 0.1 N HNO₃ then water |
| **Pump pressure fluctuation > ±5 bar** | Air bubble in pump check valves; worn piston seal | Purge pump with methanol (prime); replace piston seals ($50–150/set) |
| **ChemStation "instrument not responding"** | GPIB/LAN cable; firmware mismatch after module swap | Power-cycle in order: modules first, then PC; verify cable seating; check Agilent LabAdvisor diagnostic |
| **Peak area inflation vs standard** | Sample carryover from previous injection | Add methanol needle wash between injections; reduce sample concentration |
| **Peak area deflation vs standard** | Incomplete dissolution in sample diluent; oil still in suspension | Sonicate longer; centrifuge vial before aspiration; re-prep with LLE |
| **New column, plates too low (N < 2000 on suitability)** | Column damaged in shipping; guard cartridge inserted backward | Confirm flow direction (arrow on column); replace column; contact vendor |

Cross-reference: [`../05-home-lab-setup/failure-modes.md`](../05-home-lab-setup/failure-modes.md) for the broader catalog including operator-error failure modes.

---

## 10. Worked example

**Sample:** 40 mg/mL estradiol valerate in MCT oil, gray-market vendor vial, 5 mL. Label claim 40 mg/mL; batch number XV-25-0419.

**Prep:** Per §5.2, dilute-and-shoot in methanol, 1:1000 total → target 40 µg/mL.

**Calibration curve (prepped today from fresh 1.023 mg/mL stock of USP EV RS lot 2024-G):**

| Level | Concentration (µg/mL) | Peak area (counts) |
|---|---|---|
| 50% | 20.0 | 623,400 |
| 80% | 32.0 | 998,800 |
| 100% | 40.0 | 1,250,000 |
| 120% | 48.0 | 1,498,200 |
| 150% | 60.0 | 1,877,100 |

Linear regression: **Area = 31,250 × [EV, µg/mL] + 0 (forced)**. R² = 0.99982.

Back-calculation residuals: 50% = −0.3%, 80% = +0.1%, 100% = 0.0%, 120% = −0.1%, 150% = +0.2%. All within ±2% — curve accepted.

**System suitability (5 replicate injections of 40 µg/mL standard):**
- Mean area: 1,250,000 counts
- RSD of area: 0.8% (≤ 2.0% — PASS)
- Mean tR: 8.23 min (RSD 0.3%)
- Theoretical plates N: 4,850 (≥ 2000 — PASS)
- Tailing T (USP): 1.12 (≤ 2.0 — PASS)
- Resolution Rs (EV vs EC internal standard at tR 11.85): 8.2 (≥ 1.5 — PASS)

Suitability PASS. Proceed.

### 10.1 Pass case — vial potency 100.0%

Sample injection (10 µL of 1:1000 diluted prep):
- Peak tR: 8.21 min (within ±2% of 8.23 — PASS identity RT)
- DAD spectral match to EV library: 0.9997 (≥ 0.998 — PASS identity spectrum)
- Peak purity index: 998 (≥ 990 — PASS no coelution)
- Peak area: **1,250,000 counts**

Back-calc:
- [EV] in injection vial = 1,250,000 / 31,250 = **40.0 µg/mL**
- [EV] in original oil = 40.0 µg/mL × 1000 (dilution factor) = 40,000 µg/mL = **40.0 mg/mL**
- Potency = 40.0 / 40.0 × 100% = **100.0% of label claim**

Result: **PASS. Reportable as 40.0 mg/mL EV (100.0% label), identity confirmed by RT + DAD spectral match.**

### 10.2 Fail case — same vial hypothetically under-dosed

Hypothetical: same vial, but peak area reads **920,000 counts** (the other parameters — RT, spectrum, purity — unchanged, so it IS estradiol valerate, just less of it):

- [EV] in injection vial = 920,000 / 31,250 = **29.44 µg/mL**
- [EV] in original oil = 29.44 × 1000 = 29,440 µg/mL = **29.4 mg/mL**
- Potency = 29.4 / 40.0 × 100% = **73.6% of label claim**

Result: **FAIL potency (< 90%).** Report as "29.4 mg/mL (73.6% label claim) — FAIL USP injection monograph acceptance limit of 90.0–110.0%." Re-prep + re-run to confirm; if confirmed, vendor reporting is warranted.

### 10.3 Fail case — wrong ester (identity fail)

Hypothetical: same vial, peak area 1,248,000 counts, but tR = 9.58 min (not 8.21).

- tR 9.58 is outside ±2% of standard 8.23 (window 8.07–8.40 min) — **FAIL identity RT**
- DAD spectral match: 0.9996 to EV library (spectra are near-identical across all estradiol esters — the phenolic A-ring chromophore dominates)
- tR 9.58 matches **estradiol enanthate** (EEn) in the method library (§4 table)

Result: **FAIL identity — peak is not estradiol valerate.** Most likely wrong ester (EEn substituted for EV). Re-run with EEn reference standard to confirm; if confirmed, this is a significant vendor QC event. This is the exact failure mode that DAD alone cannot catch — retention time is the distinguishing feature, which is why a VWD-only instrument is insufficient for multi-ester identity work.

---

## 11. USP chapter citations

Used exactly as referenced:

- **USP <621> Chromatography** — general system suitability, allowable method adjustments without revalidation (column dims, particle size, flow, organic phase, pH). Harmonized November 2021 revision. Public-PDF: https://www.usp.org/sites/default/files/usp/document/harmonization/gen-chapter/harmonization-november-2021-m99380.pdf
- **USP <1225> Validation of Compendial Procedures** — defines linearity, precision, accuracy, specificity, LOD/LOQ, range, robustness for compendial analytical methods.
- **USP Estradiol Valerate monograph** — paywalled at https://online.uspnf.com. Specific method parameters cited in §2.4 and §7 above are **reconstructed from secondary sources** (Waters app note 720001985EN; peer-reviewed Bentham and Scholars Research Library HPLC method papers; USP <621> adjustment rules). Do not cite as if you read the paywalled text.
- **USP <1058> Analytical Instrument Qualification** — governs IQ/OQ/PQ of the HPLC itself. Out of scope for this SOP but required if you are building a regulated lab.

Community-practice items (not in any USP monograph):
- Bracket frequency (every 10 samples)
- 1:1000 dilution for 40 mg/mL oil → 40 µg/mL injection
- Methanol sonication of oil samples pre-filtration
- ACN freeze-precipitation for castor-oil samples
- Use of estradiol cypionate or testosterone enanthate as internal standard for EV assay

Pharmacopoeial items (reconstructed):
- Column L1 (C18) or L7 (C8), 4.6 × 150 mm × 5 µm
- Mobile phase methanol/water 80:20
- UV 280 nm (or 220 nm sensitivity variant)
- System suitability: replicate RSD ≤ 2.0% (USP <621> general); N ≥ 2000, T ≤ 2.0, Rs ≥ 1.5 (monograph-specific floors)
- Acceptance 90.0–110.0% label claim

**USP <621> method-adjustment limits for LC (corrected in round 2, R2-M1):**
- Column L and ID: L/dp constant or ± 25% to +50% (equivalently, N within −25% to +50%)
- Particle size: not freely interchangeable within L-category; must satisfy the L/dp rule
- Flow rate: ± 50%
- Mobile-phase minor component: ± 30% relative, not exceeding ± 10% absolute
- pH of aqueous buffer: ± 0.2
- Column temperature: ± 10 °C
- Injection volume: adjust to keep detector response within linearity

---

## Sources

- Agilent 1100 module reference manuals and listings: https://conquerscientific.com/wp-content/uploads/2022/10/agilent-1100-series-quaternary-pump-g1311a_manual.pdf ; https://www.labx.com/item/agilent-hp-1100-hplc-chemstation-g1322a-g1311a-g1315b/14519844 ; https://americanlaboratorytrading.com/lab-equipment-products/agilent-1100-series-hplc-system-including-g1379a-degasser-g1311a-quat-pump-g1313a-als-g1316a-colcom-g1315b-dad-5726/ (all retrieved 2026-04-23)
- Agilent ChemStation B.04.03 community discussion (EOL, Windows compat): https://community.agilent.com/technical/software/f/forum/3667/chemstation-b-04-03 ; https://www.chromforum.org/viewtopic.php?t=26656 (2026-04-23)
- ChemStation B.04.03 site prep: https://www.agilent.com/cs/library/sitepreparationchecklists/PCforChemStation_Elite8000_Win7.pdf (2026-04-23)
- Waters Symmetry C18 catalog (WAT045905 4.6×150mm 5µm; WAT054275 4.6×250mm 5µm): https://www.waters.com/nextgen/us/en/shop/columns/wat045905-symmetry-c18-column-100a-5--m-46-mm-x-150-mm-1-pk.html ; https://www.waters.com/nextgen/us/en/shop/columns/wat054275-symmetry-c18-column-100a-5--m-46-mm-x-250-mm-1-pk.html (2026-04-23)
- Agilent ZORBAX Eclipse Plus C18 (959993-902, 4.6×150mm 5µm): https://www.netascientific.com/lc-c1-columns/959993-902 ; https://chromtech.com/zorbax-eclipse-plus/ ; https://www.agilent.com/cs/library/specifications/public/820114-002.pdf (2026-04-23)
- Phenomenex SecurityGuard AJ0-4287: https://www.phenomenex.com/part?partNo=AJ0-4287 ; holder KJ0-4282 (2026-04-23)
- Waters application note 720001985EN "Analysis of Estradiol USP Method Using XBridge C18" (2007): https://www.waters.com/nextgen/us/en/library/application-notes/2007/analysis-of-estradiol-usp-method-using-xbridge-c18.html (metadata retrieved; full content retrieval timed out 2026-04-23)
- Validated RP-HPLC for estradiol valerate (Scholars Research Library): https://www.scholarsresearchlibrary.com/articles/validated-rphplc-method-for-the-determination-of-estradiol-valerate-inbulk-and-pharmaceutical-formulations.pdf (2026-04-22, via Round 1)
- Related substances in estradiol valerate (Bentham, Zorbax Eclipse XDB C8 gradient): https://www.eurekaselect.com/article/85856 (2026-04-22, via Round 1)
- USP Reference Standards catalog: USP Estradiol Valerate RS #1254009 ($297/100 mg), USP Estradiol Cypionate RS #1252003 ($277/200 mg), USP Testosterone Enanthate RS #1648004 — https://store.usp.org/ ; Round-1 snapshot at [`../_sources/usp-reference-standards-pricing-2026-04-22.md`](../_sources/usp-reference-standards-pricing-2026-04-22.md)
- USP <621> harmonized chromatography chapter (Nov 2021): https://www.usp.org/sites/default/files/usp/document/harmonization/gen-chapter/harmonization-november-2021-m99380.pdf (2026-04-23)
- USP <621> interpretation: https://www.chromatographyonline.com/view/what-the-new-usp-chapter-621-guidelines-mean-for-your-hplc-analysis (2026-04-22)
- USP-NF paywalled primary source: https://online.uspnf.com (not verified in this pass)
- Round-1 consumables pricing snapshot: [`../_sources/consumables-pricing-2026-04-22.md`](../_sources/consumables-pricing-2026-04-22.md)
- Round-1 HPLC deep-dive: [`../02-analytical-chemistry/hplc.md`](../02-analytical-chemistry/hplc.md)
- Round-1 oil-prep detail: [`../08-interpretation-qc/oil-formulation-sample-prep.md`](../08-interpretation-qc/oil-formulation-sample-prep.md)
- Round-1 failure-modes catalog: [`../05-home-lab-setup/failure-modes.md`](../05-home-lab-setup/failure-modes.md)

---

**Document status:** Round 2 deep-dive SOP. Intended as a working lab document for a refurb Agilent 1100 + USP EV RS + L1 C18 column build. Not a GLP-compliant substitute for a real method-validation protocol. Revalidate quarterly; re-verify paywalled USP monograph conditions annually or at any USP-NF revision bulletin.